Antibodies specific for immunoglobulin-like transcript 3 (ilt3) and uses thereof

ABSTRACT

Humanized, non-promiscuous monoclonal antibodies specific for immunoglobulin-like transcript 3 (ILT3), also known as Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4), are described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Pat. No.11,111,297 issued Sep. 7, 2021, which claims benefit of U.S. ProvisionalPatent Application No. 62/587,604 filed Nov. 17, 2017, each of which isherein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “24530_US_NP_SEQTXT_05NOVEMBER2018.txt”, creation date of Nov.5, 2018, and a size of 376 Kb. This sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention provides non-promiscuous monoclonal antibodiesspecific for immunoglobulin-like transcript 3 (ILT3), an inhibitoryreceptor expressed on the surface of myeloid immune cells.

(2) Description of Related Art

Immunoglobulin-like transcript 3 (ILT3), designated CD85k and also knownas Leukocyte Immunoglobulin-Like Receptor subfamily B member 4 (LILRB4)and Leukocyte Immunoglobulin-like Receptor 5 (LIR-5), is a type Imembrane protein that contains cytoplasmic immunoreceptor tyrosine-basedinhibition motif (ITIM) motifs and is involved in the down-regulation ofimmune responses (Cella et al., J Exp Med. 185 (10): 1743-51 (1997);Samaridis et al., Eur J Immunol. 27 (3): 660-665 (1997). Expression ofILT3 is up-regulated on tolerogenic dendritic cells. This gene is amember of the leukocyte immunoglobulin-like receptor (LIR) family, whichis found in a gene cluster at chromosomal region 19q13.4. The encodedprotein belongs to the subfamily B class of LIR receptors, which containtwo or four extracellular immunoglobulin domains, a transmembranedomain, and two to four ITIMs.

ILT3 is selectively expressed by myeloid antigen presenting cells (APCs)such as monocytes, macrophages, and dendritic cells, e.g.,monocyte-derived dendritic cells differentiated in the presence of IL-10or vitamin D₃. ILT3 consists of 447 amino acids with a predictedmolecular mass of about 47 kD. The amino terminal portion of ILT3 beginswith a hydrophobic signal peptide of 23 amino acids followed by anextracellular domain composed of two C₂ type immunoglobulin superfamilydomains and having the amino acid sequence set forth in SEQ ID NO: 1less the C-terminal His Tag. (The Rhesus monkey ILT3 extracellulardomain has the amino acid sequence set forth in SEQ ID NO: 2). Theputative transmembrane domain of ILT3 consists of 21 amino acids,followed by a long cytoplasmic region of 167 amino acids, which ischaracterized by the presence of motifs spaced by 26 amino acid residuesand are reminiscent of the ITIM motifs identified in KIRs(natural-killer cell Ig receptors) as binding sites for protein tyrosinephosphatase SHP-1. ILT3 is expressed on immune cells where it binds toMHC class I molecules on antigen-presenting cells and transduces anegative signal that inhibits stimulation of an immune response. Thereceptor can also function in antigen capture and presentation. ILT3 isthought to control inflammatory responses and cytotoxicity to help focusthe immune response and to limit auto-reactivity. Multiple transcriptvariants encoding different isoforms of ILT3 have been identified.

Patent publications that disclose use of an antibody for modulating ILT3activity with applications for inhibiting transplant rejection or foruse in treatments for cancer or infectious diseases include U.S. Pub.Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and, Intl.Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641,WO2016127427, WO2018089300, and WO2018148494. Of interest is Intl. Pub.No. WO2017015227, which discloses CD166, also known as lymphocyte celladhesion molecule (ALCAM), as a ligand for ILT3 and provides methods fortreating cancer comprising in some embodiments an antibody against CD166or ALCAM. Also of interest are U.S. Pat. Nos. 7,777,008 and 8,901,281,which disclose monoclonal antibody 9B11 for use in various treatmentswhere it is desirable to upregulate the immune system for anti-cancertreatments and to downregulate the immune system for inhibitingtransplant rejection.

While the patent publications disclose anti-ILT3 antibodies, in someinstances no specific antibody is disclosed or specific antibodies aredisclosed, which in some cases are shown to be promiscuous andcross-react with one or more ILT3-related receptors such as LILRA6 andILT8. Promiscuous anti-ILT3 antibodies may have off-target effects,which may have undesirable effects that contraindicate its use fortherapeutic applications. Therefore there is a need for antibodies andantigen binding fragments that specifically bind ILT3 and have nomeasurable promiscuity towards other related receptors.

BRIEF SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies and antigen bindingfragments that bind specifically to immunoglobulin-like transcript 3(ILT3) with no measurable binding to closely related proteins (e.g.,ILT5, ILT7, ILT8, or ILT11) as determined by (i) a cell ELISA using 10μg/mL antibody or antigen binding fragment or (ii) Biacore using 10μg/mL antibody or antigen binding fragment. In particular embodiments,the antibodies and antigen binding fragments specifically bind to bothhuman ILT3 and Rhesus monkey ILT3. These antibodies and antigen bindingfragments are capable of antagonizing ILT3 activity thereby enhancingdendritic cell activation and T cell priming. Tolerized dendritic cellsand myeloid-derived suppressor cells (MDSCs) are also responsive tothese antibodies. Furthermore, in vivo studies of these antibodies inhumanized NSG™ mouse model systems (The Jackson Laboratories, BarHarbor, Me.) show that these antibodies may have the ability to reducetumor burden and shift cellular phenotypes to a more activated state.

In clinical trial samples, ILT3 expression, like PD-L1, LAG3, and theGEP signature, was found to be associated with responsiveness to theanti-PD-1 antibody, pembrolizumab. Soluble ILT3 in circulation is alsoincreased in certain cancer types. Taken together, the anti-ILT3antibodies of the present invention may be useful for treatingparticular cancers either as a monotherapy treatment or in combinationwith an anti-PD-1 and/or anti-PD-L1 antibody to enhance responsivenessto the anti-PD-1 or anti-PD-L1 antibody, particularly in cancertreatments in which the cancer is non-responsive to anti-PD-1 oranti-PD-L1 monotherapies. In particular embodiments, the presentinvention provides chimeric or humanized anti-ILT3 antibodies. Incertain embodiments, the antibodies may be fully human antibodies thatcompete with the antibodies disclosed herein for binding to the ILT3epitope disclosed herein.

The present invention provides an antibody or antigen binding fragmentcomprising one, two, or three complementarity determining regions (CDRs)of a heavy chain variable V_(H) domain having heavy chaincomplementarity determining region (HC-CDR) 1, 2, and 3 and one, two, orthree CDRs of a light chain variable domain V_(L) having LC-CDR1, 2, and3, wherein the antibody or antigen binding fragment is capable ofspecifically binding human ILT3 wherein the binding of the antibody orantigen binding fragment may be determined by cell ELISA or Biacore.

In a further embodiment, the antibody or antigen binding fragment bindsto an epitope on the human ILT3 or competes with an antibody disclosedfor binding to an epitope on the human ILT3, wherein the epitopecomprises at least one amino acid within one or more of the amino acidsequences set forth in the group consisting of SEQ ID NOs:3, 4, 5, 6, 7,and 8. In further embodiments, the antibody or antigen binding fragmentbinds to an epitope on the human ILT3 or competes with an antibodydisclosed for binding to an epitope on the human ILT3, wherein theepitope comprises one or more of the amino acid sequences set forth inthe group consisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In furtherembodiments, the antibody or antigen binding fragment binds to anepitope on the human ILT3 or competes with an antibody disclosed forbinding to an epitope on the human ILT3, wherein the epitope comprisesthe amino acid sequences set forth in the group consisting of SEQ IDNOs:3, 4, 5, 6, 7, and 8. In particular embodiments, the epitope isdetermined by hydrogen deuterium exchange mass spectrometry (HDX-MS)analysis.

The present invention further provides an antibody or antigen bindingfragment that binds human ILT3 comprising a heavy chain (HC) wherein theheavy chain variable domain (V_(H)) comprises a heavy chaincomplementarity determining region (HC-CDR) 3 having an amino acidsequence selected from the group consisting of SEQ ID NO: 22, 49, 57,65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has3, 2, or 1 differences with an amino acid sequence selected from thegroup consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105.In some embodiments the amino acid sequence differences are conservativechanges/substitutions. In particular embodiments, the antibody orantigen binding fragment that binds human ILT3 comprises a heavy chain(HC) wherein the heavy chain variable domain (V_(H)) comprises a heavychain complementarity determining region (HC-CDR) 3 having an amino acidsequence selected from the group consisting of SEQ ID NO: 23, 49, 57,65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has3, 2, or 1 differences with an amino acid sequence selected from thegroup consisting of SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97, and 105.In particular embodiments the amino acid sequence differences areconservative changes/substitutions.

In a further embodiment, the antibody or antigen binding fragment bindsto an epitope on the human ILT3 or competes with an antibody disclosedfor binding to an epitope on the human ILT3, wherein the epitopecomprises at least one amino acid from one or more of the amino acidsequences set forth in in the group consisting of SEQ ID NO: 3, 4, 5, 6,7, and 8. In further embodiments, the antibody or antigen bindingfragment binds to an epitope on the human ILT3 or competes with anantibody disclosed for binding to an epitope on the human ILT3, whereinthe epitope comprises one or more of the amino acid sequences set forthin SEQ ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the antibodyor antigen binding fragment binds to an epitope on the human ILT3 orcompetes with an antibody disclosed for binding to an epitope on thehuman ILT3, wherein the epitope comprises the amino acid sequences setforth in SEQ ID NOs:3, 4, 5, 6, 7, and 8. In particular embodiments, theepitope is determined by hydrogen deuterium exchange mass spectrometry(HDX-MS) analysis.

The present invention further provides an antibody or antigen bindingfragment that binds human ILT3 comprising (a) an HC having a variabledomain (V_(H)) comprising a variable domain complementarity determiningregion (HC-CDR) 1 having the amino acid sequence set forth in SEQ ID NO:17, 47, 55, 63, 71, 79, 87, 95, or 103; an HC-CDR2 having the amino acidsequence set forth in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104;and an HC-CDR3 having the amino acid sequence set forth in SEQ ID NO:23, 49, 57, 65, 73, 81, 89, 97, or 105; and, variants thereof whereinone or more of the HC-CDRs has one, two, or three amino acidsubstitutions, additions, deletions, or combinations thereof; and (b) alight chain (LC) having variable domain (V_(L)) comprising a variabledomain complementarity determining region (LC-CDR) 1 having the aminoacid sequence set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98, or106; an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO:43, 51, 59, 67, 75, 83, 91, 99, or 107; and an LC-CDR3 having the aminoacid sequence set forth in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100, or108; and, variants thereof wherein one or more of the LC-CDRs has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof. In particular embodiments the amino acid sequencedifferences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment,HC-CDR1 has the amino acid sequence set forth in SEQ ID NO:17; HC-CDR2has the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21;HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; andLC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 34, 35, 36,37, 38, 39, 40, 41, or 42; LC-CDR2 has the amino acid sequence set forthin SEQ ID NO: 43; and, LC-CDR3 has the amino acid sequence set forth inSEQ ID NO:44; and, variants thereof wherein one or more of the HC-CDRsand LC-CDRs has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof. In particular embodiments the aminoacid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment,HC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 17; HC-CDR2has the amino acid sequence set forth in SEQ ID NO: 20; and HC-CDR3 hasthe amino acid sequence set forth in SEQ ID NO: 23; and LC-CDR1 havingthe amino acid sequence set forth in SEQ ID NO: 41; LC-CDR2 having theamino acid sequence set forth in SEQ ID NO: 43; and, LC-CDR3 having theamino acid sequence set forth in SEQ ID NO: 44; and, variants thereofwherein one or more of the HC-CDRs and LC-CDRs has one, two, or threeamino acid substitutions, additions, deletions, or combinations thereof.In particular embodiments the amino acid sequence differences areconservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, theantibody or antigen binding fragment comprises (a) a V_(H) having aframework selected from the group consisting of human V_(H)1, V_(H)2,V_(H)3, V_(H)4, V_(H)5, and V_(H)6 family and variants thereof having 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof; and, (b) a V_(L) having a frameworkselected from the group consisting of human V_(κ)1, V_(κ)2, V_(κ)3,V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6,V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10 family and variants thereof having1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof. In particular embodiments the aminoacid sequence differences are conservative changes/substitutions.

In particular embodiments, the antibody or antigen binding fragmentcomprises (a) a V_(H) having a human V_(H)1 family framework or variantthereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof; and, (b) aV_(L) having a human V_(κ)5 family framework or variant thereof having1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof. In particular embodiments the aminoacid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody, the antibody comprises a humanIgG1, IgG2, IgG3, or IgG4 HC constant domain or variant thereof having1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof compared to the amino acid sequenceof the native human IgG1, IgG2, IgG3, or IgG4 isotype HC constantdomain. In particular aspects, the constant domain may comprise aC-terminal lysine or may lack a C-terminal lysine or a C-terminalglycine-lysine dipeptide.

In particular embodiments, the heavy chain constant domain is of thehuman IgG1 isotype, which has been modified to have reduced or minimaleffector function. In further aspects, the minimal effector functionresults from an effector-less Fc mutation, which may comprise or consistof the mutation N297A or D265A/N297A as identified using Kabat numberingin which case the minimal effector function results from aglycosylation(see for example, the amino acid sequence shown in SEQ ID NO: 211wherein the N297A mutation corresponds to amino acid position 180; aD265A mutation, if present, would correspond to amino acid position148). In particular aspects, the IgG1 has been modified to comprise orconsist of an L234A, an L235A, and a D265S mutation as identified usingKabat numbering to render the Fc effector-less (see for example theamino acid sequence shown in SEQ ID NO: 12 or 13 wherein the L234A,L235A, and D265S mutations correspond to amino acid positions 117, 118,and 148, respectively).

In a further aspect, the HC constant domain is of the human IgG4 isotypeand which isotype further includes a substitution of the serine residueat position 228 (EU numbering) with proline, which corresponds toposition 108 of SEQ ID NO: 9 or 10 (Serine at position 108).

In a further embodiment of the antibody or antigen binding fragment, theantibody comprises a human kappa or lambda LC constant domain or variantthereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof compared tothe amino acid sequence of the native human kappa or lambda LC constantdomain. In particular embodiments the amino acid sequence differencesare conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, theantibody comprises (i) a V_(H) having a framework selected from thehuman V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, and V_(H)6 family and ahuman IgG1 or IgG4 HC constant domain or variant thereof comprising 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof compared to the amino acid sequenceof the native human IgG1 or IgG4 isotype HC constant domain; and, (ii)and a V_(L) having a framework selected from the human V_(κ)1, V_(κ)2,V_(κ)3, V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5,V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10 family and a human kappa orlambda LC constant domain or variant thereof comprising 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof compared to the amino acid sequence of the nativehuman kappa or lambda LC constant domain. In particular embodiments theamino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, theantibody comprises (i) a V_(H) having a human V_(H)2 family frameworkand a V_(L) having a human V_(κ)5 family framework; (ii) a human IgG1 orIgG4 HC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof compared to the amino acid sequence of the nativehuman IgG1 or IgG4 isotype HC constant domain; and, (iii) a human kappaLC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acid substitutions, additions, deletions, or combinationsthereof compared to the amino acid sequence of the native human kappa LCconstant domain. In particular embodiments the amino acid sequencedifferences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, theantibody comprises (i) a V_(H) having a human V_(H)1 family frameworkand a human V_(L) having a human V_(κ)5 family framework; (ii) a humanIgG4 HC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof compared to the amino acid sequence of the nativehuman IgG4 isotype HC constant domain; and, (iii) a human kappa LCconstant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acid substitutions, additions, deletions, or combinationsthereof compared to the amino acid sequence of the native human kappa LCconstant domain. In particular embodiments the amino acid sequencedifferences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, theantibody or antigen binding fragment comprises a V_(H) and a V_(L)having the amino acid sequences set forth in SEQ ID NO: 15 and SEQ IDNO: 16, respectively; SEQ ID NO: 45 and SEQ ID NO: 46, respectively; SEQID NO: 53 and SEQ ID NO: 54, respectively; SEQ ID NO: 61 and SEQ ID NO:62, respectively; SEQ ID NO: 69 and SEQ ID NO: 70, respectively; SEQ IDNO: 77 and SEQ ID NO: 78, respectively; SEQ ID NO: 85 and SEQ ID NO: 86,respectively; SEQ ID NO: 93 and SEQ ID NO: 94, respectively; or SEQ IDNO: 101 and SEQ ID NO: 102, respectively.

In a further embodiment of the antibody or antigen binding fragment, theantibody or antigen binding fragment comprises a V_(H) having the aminoacid sequence set forth in SEQ ID NO: 117, 118, 119, 123, 124, or 125and a V_(L) having the amino acid sequence set forth in SEQ ID NO: 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or141.

In a further embodiment of the antibody or antigen binding fragment, theantibody or antigen binding fragment comprises a V_(H) having the aminoacid sequence set forth in SEQ ID NO: 118 and a V_(L) having the aminoacid sequence set forth in SEQ ID NO: 140.

In a further embodiment of the antibody, the antibody comprises an HCconstant domain comprising the amino acid sequence set forth in SEQ IDNO: 9, 10, 11, 12, or 13. In particular aspects, the HC constant domaincomprising the amino acid sequence set forth in SEQ ID NOs: 9, 11, 12,or 13 may lack a C-terminal lysine or a C-terminal glycine-lysinedipeptide. In particular embodiments, the HC constant domain comprisesthe amino acid sequence set forth in SEQ ID NO: 10.

In a further embodiment of the antibody, the antibody comprises an LCconstant domain comprising the amino acid sequence set forth in SEQ IDNO: 14.

In a further embodiment of the antibody, the antibody comprises an HCcomprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148,149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184,185, 186, 187, 191, 192, or 193. In particular aspects, the HCcomprising the amino acid sequence set forth in SEQ ID NOs: 142, 143,144, 148, 149, 150, 167, 168, 169, 170, 174, or 175, may lack aC-terminal lysine or a C-terminal glycine-lysine dipeptide. Inparticular embodiments, the HC comprises the amino acid sequence setforth in SEQ ID NO: 143 or 177. In particular embodiments, the HC setforth in SEQ ID NO: 177 further lacks a C-terminal glycine.

In a further embodiment of the antibody, the antibody comprises an LCcomprising the amino acid sequence set forth in SEQ ID NO: 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166.In particular embodiments, the LC comprises the amino acid set forth inSEQ ID NO: 165.

In a further embodiment of the antibody, the antibody comprises an HChaving the amino acid sequence set forth in SEQ ID NO:143 and an LCcomprising the amino acid sequence set forth in SEQ ID NO:165. Inparticular aspects, the HC comprising the amino acid sequence set forthin SEQ ID NO: 143 lacks a C-terminal lysine or a C-terminalglycine-lysine dipeptide.

The present invention further provides a chimeric, humanized, orrecombinant human antibody or antigen binding fragment that binds to anepitope on a human ILT3, wherein the epitope comprises at least oneamino acid within the amino acid sequences set forth in the groupconsisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In a further embodiment,the chimeric, humanized, or recombinant human antibody or antigenbinding fragment binds to an epitope on a human ILT3 comprising theamino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. Inthese embodiments, the epitope is determined by hydrogen deuteriumexchange mass spectrometry (HDX-MS) analysis.

The present invention further provides a chimeric, humanized, orrecombinant human antibody or antigen binding fragment that binds ILT3wherein the binding cross-blocks or competes with the binding of anantibody comprising a heavy chain having the amino acid sequence setforth in SEQ ID NO: 15 and a light chain having the amino acid sequenceshown in SEQ ID NO: 16. In a further embodiment, the chimeric,humanized, or recombinant human antibody or antigen binding fragmentthat cross-blocks or competes with an antibody comprising a heavy chainhaving the amino acid sequence set forth in SEQ ID NO: 15 and a lightchain having the amino acid sequence shown in SEQ ID NO: 16 binds anepitope on ILT3 that comprises the amino acid sequences set forth in SEQID NOS: 3, 4, 5, 6, 7, and 8.

The present invention further provides a composition comprising one ormore of any one of the antibody or antigen binding fragment disclosed orclaimed herein and a pharmaceutically acceptable carrier.

The present invention further provides a method for treating a cancer ina subject comprising administering to the subject an effective amount ofan antibody or antigen binding fragment disclosed or claimed hereinsufficient to treat the cancer in the subject.

In a further embodiment, the cancer is pancreatic cancer, melanomas,breast cancer, lung cancer, head and neck cancer, bronchus cancer,colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer,ovarian cancer, urinary bladder cancer, brain or central nervous systemcancer, peripheral nervous system cancer, esophageal cancer, cervicalcancer, uterine or endometrial cancer, cancer of the oral cavity orpharynx, liver cancer, kidney cancer, testicular cancer, biliary tractcancer, small bowel or appendix cancer, salivary gland cancer, thyroidgland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, orcancer of hematological tissues.

The present invention further provides a method for treatment of acancer in a subject comprising administering to the subject concurrentlyor consecutively an antibody or antigen binding fragment disclosedherein in combination with one or more inhibitors or antagonists ofPD-1, PD-L1 and/or PD-L2. In one embodiment, the antagonist of PD-1 isan antibody or antigen binding fragment thereof that binds to human PD-1and blocks the binding of PD1 to human PD-L1 and PD-L2. In oneembodiment, the antagonist of PD-L1 or PD-L2 is an antibody or antigenbinding fragment thereof that binds to human PD-L1 or PD-L2 and blocksthe binding of human PD-L1 or PD-L2 PD1.

In a further embodiment, the anti PD1 antagonist is an anti-PD-1antibody is nivolumab, pembrolizumab, cemiplimab, or pidilizumab and thePD-L1 inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70,MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

The present invention further provides an antibody or antigen bindingfragment disclosed or claimed herein for treatment of cancer in asubject.

In a further embodiment, the cancer is pancreatic cancer, melanomas,breast cancer, lung cancer, head and neck cancer, bronchus cancer,colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer,ovarian cancer, urinary bladder cancer, brain or central nervous systemcancer, peripheral nervous system cancer, esophageal cancer, cervicalcancer, uterine or endometrial cancer, cancer of the oral cavity orpharynx, liver cancer, kidney cancer, testicular cancer, biliary tractcancer, small bowel or appendix cancer, salivary gland cancer, thyroidgland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, orcancer of hematological tissues.

The present invention further provides an antibody or antigen bindingfragment disclosed or claimed herein for treatment of a cancer in asubject wherein the treatment further comprises one or more inhibitorsor antagonists of PD-1, PD-L1 and/or PD-L2.

In one embodiment, the antagonist of PD-1 is an antibody or antigenbinding fragment thereof that binds to human PD-1 and blocks the bindingof PD1 to PD-L1 and PD-L2.

In one embodiment, the antagonist of PD-L1 or PD-L2 is an antibody orantigen binding fragment thereof that binds to human PD-L1 or PD-L2 andblocks the binding of human PD-L1 or PD-L2 PD1.

In a further embodiment, the anti-PD-1 antibody is nivolumab,pembrolizumab, cemiplimab, or pidilizumab and the PD-L1 inhibitor isdurvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736,MSB-0010718C, or MDX-1105.

The present invention further provides for use of an antibody or antigenbinding fragment disclosed or claimed herein for the treatment of acancer.

The present invention further provides for use of an antibody or antigenbinding fragment disclosed or claimed herein for the manufacture of amedicament for the treatment of a cancer.

In a further embodiment, the cancer is pancreatic cancer, melanomas,breast cancer, lung cancer, head and neck cancer, bronchus cancer,colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer,ovarian cancer, urinary bladder cancer, brain or central nervous systemcancer, peripheral nervous system cancer, esophageal cancer, cervicalcancer, uterine or endometrial cancer, cancer of the oral cavity orpharynx, liver cancer, kidney cancer, testicular cancer, biliary tractcancer, small bowel or appendix cancer, salivary gland cancer, thyroidgland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, orcancer of hematological tissues.

The present invention further provides a composition comprising any oneof the aforementioned antibodies or antigen binding fragments and apharmaceutically acceptable carrier. In particular embodiments, thecomposition comprises a mixture of antibodies comprising a heavy chainhaving a C-terminal lysine and antibodies comprising a heavy chainlacking a C-terminal lysine. In particular embodiments, the compositioncomprises an antibody disclosed herein wherein the predominant antibodyform comprises a heavy chain having a C-terminal lysine. In particularembodiments, the composition comprises an antibody disclosed hereinwherein the predominant antibody form comprises a heavy chain lacking aC-terminal lysine. In particular embodiments, the composition comprisesan antibody disclosed herein wherein about 100% of the antibodies in thecomposition comprise a heavy chain lacking a C-terminal lysine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, and FIG. 1F show acomparison of the selectivity of several of the anti-ILT3 antibodiesdisclosed herein to monoclonal antibody 9B11 and mouse IgG1 (mIGgG1)using a cell-based ELISA format. CHO-K1 cells expressing human ILT3(FIG. 1A), Rhesus monkey ILT3 (FIG. 1B), human ILT5 (FIG. 1C), humanILT7 (FIG. 1D), human ILT8 (FIG. 1E), or human ILT11 (FIG. 1F) were eachtested with monoclonal antibody p40B5 (LB179.40B5.1A1), p49C6(LB181.49C6.1A1), and p52B8 1b181.52B8.1B1); antibody 9B11 (U.S. Pat.No. 7,777,008 as having the amino acid sequences of SEQ ID NO: 33 (lightchain) and SEQ ID NO: 34 (heavy chain)), and mouse IgG1.

FIG. 2A shows data characteristics on binding affinity, isoelectricpoint, purity of monomer species, and thermal stability measurements forvariants of mAb 10. Terms: “huILT3” refers to human ILT3; “rhILT3”refers to Rhesus monkey ILT3; “pI” refers to isoelectric point; “Tm”refers to temperature mid-point of a thermal unfolding curve; “Tagg”refers to mid-point of a thermal aggregation curve; “SEC” referssize-exclusion ultra-high performance liquid chromatography).

FIG. 2B shows the relationship of SEC purity and melting temperature ofhumanized light chain variants of mAb 10 (M64V VH1 IgG4). VL1-VL8 referto variants having the amino acid sequence set forth in SEQ ID NOs:126-133, respectively.

FIG. 3A shows a deuterium labeling difference heatmap of the human ILT3extracellular domain amino acid residues that are bound by ChimericAnti-ILT3 52B8 mouse 52B8 VH parental/human IgG4 (S228P): mouse 52B8parental VL/human Kappa antibody (“c58B2”; mAb 73). These six peptidedomains, which comprise the epitope bound by the antibody (residues18-23 (ISWGNS; SEQ ID NO: 3), residues 64-69 (IPSMTE; SEQ ID NO: 4),residues 96-101 (MTGAYS; SEQ ID NO: 5), residues 124-131 (QSRSPMDT; SEQID NO: 6), residues 152-159 (AQQHQAEF; SEQ ID NO: 7) and residues184-187 (LLSH; SEQ ID NO: 8)), are located near the border of the D1 andD2 domains of the ILT3 extracellular domain. The amino acid sequence ofhuman extracellular domain with C-terminal His Tag is set forth in SEQID NO: 1.

FIG. 3B shows a first view and a second view of a surface structuremodel of the extracellular domain of human ILT3. The dark region of themodel shows the location of the six peptide domains comprising the humanILT3-His epitope bound by c58B8 (mAb 73).

FIG. 3C is a ribbon diagram showing the placement of the epitope on theILT3 extracellular domain: ISWGNS (SEQ ID NO: 3), IPSMTE (SEQ ID NO: 4),MTGAYS (SEQ ID NO: 5), QSRSPMDT (SEQ ID NO: 6), AQQHQAEF (SEQ ID NO: 7)and LLSH (SEQ ID NO: 8).

FIG. 3D shows a deuterium labeling difference heatmap of the human ILT3extracellular domain amino acid residues that are bound by antibodyZM4.1.

FIG. 3E shows a deuterium labeling difference heatmap of the human ILT3extracellular domain amino acid residues that are bound by antibodyDX446.

FIG. 3F shows a deuterium labeling difference heatmap of the human ILT3extracellular domain amino acid residues that are bound by antibodyDX439.

FIG. 3G shows a deuterium labeling difference heatmap of the human ILT3extracellular domain amino acid residues that are bound by antibody9B11.

FIG. 4 shows free c52B8 (mAb 73) concentrations in blood after multipledoses in humanized tumor models (Panc08.13 and SK-MEL-5). Free c52B8concentrations are expressed by circles and squares. Dashed linesindicate simulated historical antibody levels after IV bolusadministration of 1, 3, 10, or 30 mg/kg of humanized IgG4 in C57BL/6Jmice.

FIG. 5A shows a human dendritic cell (DC) functional assay demonstratinganti-ILT3 antibody chimeric antibodies in which the V_(H) and V_(L) fromp52B8 fused to IgG4 Fc (c52B8; mAb 73), IgG1 Fc (mAb 78), or IgG1(N297A) Fc (mAb 76) had comparable ability to activate dendritic cells(DCs). Human immature DCs were prepared and differentiated into CD11c+dendritic cells with GM-CSF (1000 U/mL) and IL-4 (1000 U/mL) over 5days. These cells were treated with IL-10, LPS (a gram negativebacterial cell wall component and a TLR4 ligand (Raetz et al. Ann. Rev.Biochem. 71: 635-700 (2002)), and varying concentrations of theindicated antibodies for 42 hours. The data shown are mean and s.d. oftwo technical replicates. This experiment is representative of fourindependent studies. Control IgGs had no effect (not shown).

FIG. 5B and FIG. 5C show that humanized 52B8 (lot 26AVY; mAb 46) isindistinguishable from c52B8 (mAb 73) in the human DC functional assayusing DCs from two different healthy human donors. The data shown aremean and s.d. of two technical replicates. The data shown arerepresentative of three independent studies using these two donors.

FIG. 6A and FIG. 6B show that anti-ILT3 antibody c52B8 (mAb 73) andhumanized anti-ILT3 antibody 52B8 (mAb 46; lot 26AVY) reduce suppressivecapacity of myeloid-derived suppressor cells (MDSCs). The T cellsuppression assay was conducted with a T cell to MDSC ratio of 4:1. Thedata shown are means and s.d. of three technical replicates at the levelof the T cell assay step. The experiment shown is representative of twoindependent studies using PBMCs from the same two donors withqualitatively similar results.

FIG. 7 shows c52B8 inhibits growth of SK-MEL-5 tumors in SK-MEL-5human-NSG mice bearing SK-MEL-5 subcutaneous tumors. Animals wererandomized to treatment on the basis of tumor volume on day 21post-implantation and dosed s.c. with 20 mg/kg of c52B8 or isotypecontrol once weekly beginning on day 21. Data shown in the top panel aremeans and std. error (nine per group). Individual animal tumor growthcurves are shown in the middle and bottom panels. Body weight decreasedto a similar degree in both control and 52B8 groups. This study isrepresentative of three independent studies.

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show the effect of c52B8 in tumorgrowth and immune activation in SK-MEL-5 hu-NSG model. FIG. 8A shows atumor growth curve; FIG. 8B shows CyTOF quantification of TILs collected7 days after the 2^(nd) dose: % CD4⁺ T regulatory cells and CD69expression levels on CD4⁺ T cells; FIG. 8C shows sHLA-G levels in bloodplasma harvested at the end of the study; FIG. 8D shows IHC analysis ofhuman CD3⁺ T cells infiltration in the tumor, 4 tumors in each group.

FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D show the effect of a c52B8 andpembrolizumab combination in Panc 08.13 human-NSG mice. FIG. 9A shows atumor growth curve; FIG. 9B shows CyTOF quantification of % Tregs andCD69 expression levels on CD4+ T cells from tumors harvested at the endof the study; FIG. 9C shows plasma sHLA-G levels in terminal bloodsamples; FIG. 9D shows plasma IFNγ and IL-8 levels in terminal bloodsamples quantitated using 10 plex MSD (Meso Scale Discovery).

FIG. 10 shows that humanized anti-ILT3 antibody 52B8 (mAb 46) reducesthe suppressive capacity of MDSCs to an extent comparable to chimericanti-ILT3 antibody c52B8 (mAb 73) in an MDSC/T cell suppression assay ata 4:1 ratio of T cell to MDSC.

FIG. 11 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb46) and pembrolizumab combination in an MDSC/T cell suppression assay ateither a 4:1 or 8:1 ratio of T cell to MDSC using MDSC cells obtainedfrom human donor D001003835.

FIG. 12 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb46) and pembrolizumab combination in an MDSC/T cell suppression assay atan 8:1 ratio of T cell to MDSC using MDSC cells obtained from humandonor D001003180.

FIG. 13 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb46) and pembrolizumab combination in an MDSC/T cell suppression assay atan 4:1 ratio of T cell to MDSC using MDSC cells obtained from humandonor D001003507.

FIG. 14 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb46) and pembrolizumab combination in an MDSC/T cell suppression assay atan 8:1 ratio of T cell to MDSC using MDSC cells obtained from humandonor D001003428.

FIG. 15 shows the effect humanized anti-ILT3 antibody 52B8 (mAb 46) andpembrolizumab combination in a mixed lymphocyte reaction ofIL-10-polarized human monocyte-derived dendritic cells and allogenicCD8+ T cells, incubated for four days followed by measurement ofinterferon gamma (IFNγ) in the culture supernatant as a read out of Tcell activation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides non-promiscuous monoclonal antibodiesspecific for human immunoglobulin-like transcript 3 (ILT3), aninhibitory receptor expressed on the surface of myeloid immune cells.

Definitions

The term “immunoglobulin-like transcript 3” (abbreviated herein as“ILT3”, and also known as LIR-5, LILRB4, or CD85k), as used herein andunless otherwise indicated, refers to the human member of the ILT3family, which is selectively expressed by myeloid antigen presentingcells (APCs) such as monocytes, macrophages, and dendritic cells, e.g.,monocyte-derived dendritic cells differentiated in the presence of IL-10or vitamin D₃.

As used herein, “antibody” refers to an entire immunoglobulin, includingrecombinantly produced forms and includes any form of antibody thatexhibits the desired biological activity. Thus, it is used in thebroadest sense and specifically covers, but is not limited to,monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), humanized antibodies, fully human antibodies, biparatopicantibodies, humanized camelid heavy chain antibodies, andnon-human/human chimeric antibodies. “Parental antibodies” areantibodies obtained by exposure of an immune system to an antigen priorto modification of the antibodies for an intended use, such ashumanization of a non-human antibody for use as a human therapeuticantibody.

An “antibody” refers, in one embodiment, to a glycoprotein comprising atleast two heavy chains (HCs) and two light chains (LCs) inter-connectedby disulfide bonds, or an antigen binding portion thereof. Each heavychain is comprised of a heavy chain variable region or domain(abbreviated herein as V_(H)) and a heavy chain constant region ordomain. In certain naturally occurring IgG, IgD and IgA antibodies, theheavy chain constant region is comprised of three domains, C_(H)1,C_(H)2 and C_(H)3. In certain naturally occurring antibodies, each lightchain is comprised of a light chain variable region or domain(abbreviated herein as V_(L)) and a light chain constant region ordomain. The light chain constant region is comprised of one domain, CL.The human V_(H) includes six family members: V_(H)1, V_(H)2, V_(H)3,V_(H)4, V_(H)5, and V_(H)6 and the human V_(L) family includes 16 familymembers: V_(κ)1, V_(κ)2, V_(κ)3, V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2,V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10.Each of these family members can be further divided into particularsubtypes.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDR regions andfour FR regions, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant regions of the antibodies maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system. Theassignment of amino acids to each domain is, generally, in accordancewith the definitions of Sequences of Proteins of Immunological Interest,Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.;NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75;Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al.,(1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature342:878-883.

In general, while an antibody comprises six CDRs, three on the V_(H) andthree on the V_(L), the state of the art recognizes that in most cases,the CDR3 region of the heavy chain is the primary determinant ofantibody specificity, and examples of specific antibody generation basedon CDR3 of the heavy chain alone are known in the art (e.g., Beiboer etal., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998); Xu et al., Immunity 13: 37-45 (2000). See Kabat et al.(1991) Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (definingthe CDR regions of an antibody by sequence); see also Chothia and Lesk(1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of anantibody by structure).

The following general rules shown in Table 1 may be used to identify theCDRs in an antibody sequence. There are rare examples where thesevirtually constant features do not occur; however, the Cys residues arethe most conserved feature.

TABLE 1 Light chain CDR1 Start About amino acid residue 24 Residuebefore Usually a Cys Residue after Usually a Trp. Typically Trp-Tyr-Gln,but also, Trp-Leu-Gln, Trp- Phe-Gln, or Trp-Tyr-Leu Length 10 to 17amino acid residues Light chain CDR2 Start Usually 16 amino acidresidues after the end of CDR1 Residues before Generally Ile-Tyr, butalso, Val-Tyr, Ile-Lys, or Ile-Phe Length Usually seven amino acidresidues Light chain CDR3 Start Usually 33 amino acid residues after endof CDR2 Residue before Usually Cys Residues after UsuallyPhe-Gly-Xaa-Gly (SEQ ID NO: 221) Length Seven to 11 amino acid residuesHeavy chain CDR1 Start About amino acid residue 26 (usually four aminoacid residues after a Cys) [Chothia/AbM defintion]; Kabat definitionstarts five amino acid residues later Residues before UsuallyCys-Xaa-Xaa-Xaa (SEQ ID NO: 222) Residues after Usually a Trp. TypicallyTrp-Val, but also, Trp-Ile or Trp-Ala Length 10 to 12 amino acidresidues [AbM definition]; Chothia definition excludes the last fouramino acid residues Heavy chain CDR2 Start Usually 15 amino acidresidues after the end of Kabat/AbM definition) of heavy chain CDR1Residues before Typically Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 223), but anumber of variations Residues afterLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala Length Kabat definition16 to 19 amino acid residues; AbM (and recent Chothia) definition endsseven amino acid residues earlier Heavy chain CDR3 Start Usually 33amino acid residues after end of heavy chain CDR2 (usually two aminoacid residues after a Cys) Residues before Usually Cys-Xaa-Xaa(typically Cys-Ala-Arg) Residues after Usually Trp-Gly-Xaa-Gly (SEQ IDNO: 224) Length Three to 25 amino acid residues

In general, the basic antibody structural unit comprises a tetramer.Each tetramer includes two identical pairs of polypeptide chains, eachpair having one “light” chain (about 25 kDa) and one “heavy” chain(about 50-70 kDa). The amino-terminal portion of each chain includes avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The carboxy-terminal portion of theheavy chain may define a constant region primarily responsible foreffector function of the antibody. Typically, human light chains areclassified as kappa and lambda light chains. Furthermore, human heavychains are typically classified as mu, delta, gamma, alpha, or epsilon,and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989).

The heavy chain of an antibody may or may not contain a terminal lysine(K) residue, or terminal glycine and lysine (GK) residues. Thus, inparticular embodiments of the anti-ILT3 antibodies herein comprising aheavy chain constant region amino acid sequence shown herein lacking aterminal lysine but terminating with a glycine residue further includeembodiments in which the terminal glycine residue is also lacking. Thisis because the terminal lysine and sometimes glycine and lysine togethermay be cleaved during expression of the antibody or cleaved off whenintroduced into the human body with no apparent adverse effect onantibody efficacy, stability, or immunogenicity. In some cases cases,the nucleic acid molecule encoding the heavy chain may purposely omitthe codons encoding the terminal lysine or the codons for the terminallysine and glycine.

As used herein, “antigen binding fragment” refers to fragments ofantibodies, i.e. antibody fragments that retain the ability to bindspecifically to the antigen bound by the full-length antibody, e.g.fragments that retain one or more CDR regions. Examples of antibodybinding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,and Fv fragments; diabodies; single-chain antibody molecules, e.g.,scFv; nanobodies and multispecific antibodies formed from antibodyfragments.

As used herein, a “Fab fragment” is comprised of one light chain and theC_(H)1 and variable regions of one heavy chain. The heavy chain of a Fabmolecule cannot form a disulfide bond with another heavy chain molecule.A “Fab fragment” can be the product of papain cleavage of an antibody.

As used herein, a “Fab′ fragment” contains one light chain and a portionor fragment of one heavy chain that contains the V_(H) domain and theC_(H)1 domain and also the region between the C_(H)1 and C_(H)2 domains,such that an interchain disulfide bond can be formed between the twoheavy chains of two Fab′ fragments to form a F(ab′)₂ molecule.

As used herein, a “F(ab′)₂ fragment” contains two light chains and twoheavy chains containing the V_(H) domain and a portion of the constantregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond is formed between the two heavy chains. An F(ab′)₂fragment thus is composed of two Fab′ fragments that are held togetherby a disulfide bond between the two heavy chains. An “F(ab′)₂ fragment”can be the product of pepsin cleavage of an antibody.

As used herein, an “Fv region” comprises the variable regions from boththe heavy and light chains, but lacks the constant regions.

These and other potential constructs are described in Chan & Carter(2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtainedusing conventional techniques known to those with skill in the art, andthe fragments are screened for utility in the same manner as are intactantibodies. Antigen-binding portions can be produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins.

As used herein, an “Fc” region contains two heavy chain fragmentscomprising the C_(H)2 and C_(H)3 domains of an antibody. The two heavychain fragments are held together by two or more disulfide bonds and byhydrophobic interactions of the C_(H)3 domains.

As used herein, a “diabody” refers to a small antibody fragment with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementarity domainsof another chain and create two antigen-binding sites. Diabodies aredescribed more fully in, e.g., EP 404,097; WO 93/11161; and Holliger etal. (1993) Proc. Nat. Acad. Sci. USA 90: 6444-6448. For a review ofengineered antibody variants generally see Holliger and Hudson (2005)Nat. Biotechnol. 23:1126-1136.

As used herein, a “bispecific antibody” is an artificial hybrid antibodyhaving two different heavy/light chain pairs and thus two differentbinding sites. For example, a bispecific antibody may comprise a firstheavy/light chain pair comprising one heavy and one light chain of afirst antibody comprising at least the six CDRs of an anti-ILT3 antibodydisclosed herein or embodiments wherein one or more of the six CDRs hasone, two, or three amino acid substitutions, additions, deletions, orcombinations thereof along with a second heavy/light chain paircomprising one heavy and one light chain of a second antibody havingspecificity for an antigen of interest other than ILT3. Bispecificantibodies can be produced by a variety of methods including fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai, et al.,(1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) JImmunol. 148:1547-1553. In addition, bispecific antibodies may be formedas “diabodies” (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as“Janusins” (Traunecker, et al., (1991) EMBO J. 10:3655-3659 andTraunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).

As used herein, “isolated” antibodies or antigen-binding fragmentsthereof are at least partially free of other biological molecules fromthe cells or cell cultures in which they are produced. Such biologicalmolecules include nucleic acids, proteins, lipids, carbohydrates, orother material such as cellular debris and growth medium. An isolatedantibody or antigen-binding fragment may further be at least partiallyfree of expression system components such as biological molecules from ahost cell or of the growth medium thereof. Generally, the term“isolated” is not intended to refer to a complete absence of suchbiological molecules or to an absence of water, buffers, or salts or tocomponents of a pharmaceutical formulation that includes the antibodiesor fragments.

As used herein, a “monoclonal antibody” refers to a population ofsubstantially homogeneous antibodies, i.e., the antibody moleculescomprising the population are identical in amino acid sequence exceptfor possible naturally occurring mutations that may be present in minoramounts. In contrast, conventional (polyclonal) antibody preparationstypically include a multitude of different antibodies having differentamino acid sequences in their variable domains that are often specificfor different epitopes. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler et al. (1975) Nature 256: 495, or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al. (1991) Nature 352: 624-628 andMarks et al. (1991) J Mol. Biol. 222: 581-597, for example. See alsoPresta (2005) J. Allergy Clin. Immunol. 116:731.

As used herein, a “chimeric antibody” is an antibody having the variabledomain from a first antibody and the constant domain from a secondantibody wherein (i) the first and second antibodies are from differentspecies (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc.Natl. Acad. Sci. USA 81: 6851-6855) or (ii) the first and secondantibodies are from different isotypes, e.g., variable domain from anIgG1 antibody and the constant domains from an IgG4 antibody). In oneaspect, the variable domains are obtained from a non-human antibody suchas a mouse antibody (the “parental antibody”), and the constant domainsequences are obtained from a human antibody. In a further aspect, thevariable domains are humanized variable domains from a mouse antibodyand the constant domains of a human antibody.

As used herein, a “humanized antibody” refers to forms of antibodiesthat contain sequences from both human and non-human (e.g., murine, rat)antibodies. In general, the humanized antibody will comprise all of atleast one, and typically two, variable domains, in which thehypervariable loops correspond to those of a non-human immunoglobulin,and all or substantially all of the framework (FR) regions are those ofa human immunoglobulin sequence. The humanized antibody may optionallycomprise at least a portion of a human immunoglobulin constant region(Fc).

“Humanization” (also called Reshaping or CDR-grafting) is now awell-established technique for reducing the immunogenicity of monoclonalantibodies (mAbs) from xenogeneic sources (commonly rodent) and forimproving the effector functions (ADCC, complement activation, C1qbinding). The engineered mAb is engineered using the techniques ofmolecular biology, however simple CDR-grafting of the rodentcomplementarity-determining regions (CDRs) into human frameworks oftenresults in loss of binding affinity and/or specificity of the originalmAb. In order to humanize an antibody, the design of the humanizedantibody includes variations such as conservative amino acidsubstitutions in residues of the CDRs, and back substitution of residuesfrom the rodent mAb into the human framework regions (back mutations).The positions can be discerned or identified by sequence comparison forstructural analysis or by analysis of a homology model of the variableregions' 3D structure. The process of affinity maturation has mostrecently used phage libraries to vary the amino acids at chosenpositions. Similarly, many approaches have been used to choose the mostappropriate human frameworks in which to graft the rodent CDRs. As thedatasets of known parameters for antibody structures increases, so doesthe sophistication and refinement of these techniques. Consensus orgermline sequences from a single antibody or fragments of the frameworksequences within each light or heavy chain variable region from severaldifferent human mAbs can be used. Another approach to humanization is tomodify only surface residues of the rodent sequence with the most commonresidues found in human mAbs and has been termed “resurfacing” or“veneering.” Often, the human or humanized antibody is substantiallynon-immunogenic in humans.

As used herein, “non-human amino acid sequences” with respect toantibodies or immunoglobulins refers to an amino acid sequence that ischaracteristic of the amino acid sequence of a non-human mammal. Theterm does not include amino acid sequences of antibodies orimmunoglobulins obtained from a fully human antibody library wherediversity in the library is generated in silico (See for example, U.S.Pat. No. 8,877,688 or 8,691,730).

As used herein, “effector functions” refer to those biologicalactivities attributable to the Fc region of an antibody, which vary withthe antibody isotype. Examples of antibody effector functions include:Clq binding and complement dependent cytotoxicity (CDC); Fc receptorbinding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g. B cellreceptor); and B cell activation.

As used herein, “conservatively modified variants” or “conservativesubstitution” refers to substitutions of amino acids with other aminoacids having similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are set forth in Table 2.

TABLE 2 Original Conservative Original Conservative residue substitutionresidue substitution Ala (A) Gly; Ser Leu (L) Ile; Val Arg (R) Lys; HisLys (K) Arg; His Asn (N) Gln; His Met (M) Leu; Ile; Tyr Asp (D) Glu; AsnPhe (F) Tyr; Met; Leu Cys (C) Ser; Ala Pro (P) Ala Gln (Q) Asn Ser (S)Thr Glu (E) Asp; Gln Thr (T) Ser Gly (G) Ala Trp (W) Tyr; Phe His (H)Asn; Gln Tyr (Y) Trp; Phe Ile (I) Leu; Val Val (V) Ile; Leu

As used herein, the term “epitope” or “antigenic determinant” refers toa site on an antigen (e.g., ILT3) to which an immunoglobulin or antibodyspecifically binds. Epitopes within protein antigens can be formed bothfrom contiguous amino acids (usually a linear epitope) or noncontiguousamino acids juxtaposed by tertiary folding of the protein (usually aconformational epitope). Epitopes formed from contiguous amino acids aretypically, but not always, retained on exposure to denaturing solvents,whereas epitopes formed by tertiary folding are typically lost ontreatment with denaturing solvents. A contiguous linear epitopecomprises a peptide domain on an antigen comprising at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. A noncontiguousconformational epitope comprises one or more peptide domains or regionson an antigen bound by an antibody interspersed by one or more aminoacids or peptide domains not bound by the antibody, each domainindependently comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14or 15 amino acids. Methods for determining what epitopes are bound by agiven antibody (i.e., epitope mapping) are well known in the art andinclude, for example, immunoblotting and immunoprecipitation assays,wherein overlapping or contiguous peptides (e.g., from ILT3) are testedfor reactivity with a given antibody (e.g., anti-ILT3 antibody). Methodsof determining spatial conformation of epitopes include techniques inthe art and those described herein, for example, x-ray crystallography,two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identifying themolecular determinants on the antigen involved in antibody-antigenrecognition using techniques in the art and those described herein, forexample, x-ray crystallography, two-dimensional nuclear magneticresonance, and Hydrogen-Deuterium-Exchange-with-Mass-Spectroscopy(HDX-MS).

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment of aminoacid residues or combinations of segments of amino acids, as determinedby a given method. Techniques for determining whether antibodies bind tothe “same epitope on ILT3” with the antibodies described herein include,for example, epitope mapping methods, such as, x-ray analyses ofcrystals of antigen:antibody complexes, which provides atomic resolutionof the epitope, and HDX-MS. Other methods that monitor the binding ofthe antibody to antigen fragments (e.g. proteolytic fragments) or tomutated variations of the antigen where loss of binding due to amodification of an amino acid residue within the antigen sequence isoften considered an indication of an epitope component (e.g. alaninescanning mutagenesis—Cunningham & Wells (1985) Science 244:1081). Inaddition, computational combinatorial methods for epitope mapping canalso be used. These methods rely on the ability of the antibody ofinterest to affinity isolate specific short peptides from combinatorialphage display peptide libraries.

Antibodies that “compete with another antibody for binding to a targetsuch as ILT3” refer to antibodies that inhibit (partially or completely)the binding of the other antibody to the target, i.e., ILT3. Whether twoantibodies compete with each other for binding to a target, i.e.,whether and to what extent one antibody inhibits the binding of theother antibody to a target, may be determined using known competitionexperiments. In certain embodiments, an antibody competes with, andinhibits binding of another antibody to a target by at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition orcompetition may be different depending on which antibody is the“blocking antibody” (i.e., the cold antibody that is incubated firstwith the target). Competition assays can be conducted as described, forexample, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006;doi:10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by EdHarlow and David Lane, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., USA 1999. Competing antibodies bind to the same epitope,an overlapping epitope or to adjacent epitopes (e.g., as evidenced bysteric hindrance).

Other competitive binding assays include: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using 1-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, “specifically binds” refers, with respect to an antigenor molecule such as human ILT3, to the preferential association of anantibody or other ligand, in whole or part, with human ILT3 and not toother molecules, particularly molecules found in human blood or serum.Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁷ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁶ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “specifically binds” or “binds specifically” to human ILT3 refersto an antibody that binds to the human ILT3 with high affinity, whichmeans having a K_(D) of 10⁻⁷ M or less, in particular embodiments aK_(D) of 10⁻⁸ M or less, or 5×10⁻⁹ M or less, or between 10⁻⁸ M and10⁻¹¹ M or less, but does not bind with measurable binding to closelyrelated proteins such as human ILT5, human ILT7, human ILT8, and humanILT11 as determined in a cell ELISA or Biacore assay using 10 μg/mLantibody.

As used herein, an antigen is “substantially identical” to a givenantigen if it exhibits a high degree of amino acid sequence identity tothe given antigen, for example, if it exhibits at least 80%, at least90%, at least 95%, at least 97%, or at least 99% or greater amino acidsequence identity to the amino acid sequence of the given antigen. Byway of example, an antibody that binds specifically to human ILT3 mayalso cross-react with ILT3 from certain non-human primate species (e.g.,rhesus monkey or cynomolgus monkey).

As used herein, “isolated nucleic acid molecule” means a DNA or RNA ofgenomic, mRNA, cDNA, or synthetic origin or some combination thereofwhich is not associated with all or a portion of a polynucleotide inwhich the isolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty or moreother proteins or portions or fragments thereof, or may include operablylinked regulatory sequences that control expression of the coding regionof the recited nucleic acid sequences, and/or may include vectorsequences.

As used herein, “treat” or “treating” means to administer a therapeuticagent, such as a composition containing any of the antibodies or antigenbinding fragments thereof of the present invention, internally orexternally to a subject or patient having one or more disease symptoms,or being suspected of having a disease, for which the agent hastherapeutic activity or prophylactic activity. Typically, the agent isadministered in an amount effective to alleviate one or more diseasesymptoms in the treated subject or population, whether by inducing theregression of or inhibiting the progression of such symptom(s) by anyclinically measurable degree. The amount of a therapeutic agent that iseffective to alleviate any particular disease symptom may vary accordingto factors such as the disease state, age, and weight of the patient,and the ability of the drug to elicit a desired response in the subject.Whether a disease symptom has been alleviated can be assessed by anyclinical measurement typically used by physicians or other skilledhealthcare providers to assess the severity or progression status ofthat symptom. The term further includes a postponement of development ofthe symptoms associated with a disorder and/or a reduction in theseverity of the symptoms of such disorder. The terms further includeameliorating existing uncontrolled or unwanted symptoms, preventingadditional symptoms, and ameliorating or preventing the underlyingcauses of such symptoms. Thus, the terms denote that a beneficial resulthas been conferred on a human or animal subject with a disorder, diseaseor symptom, or with the potential to develop such a disorder, disease orsymptom.

As used herein, “treatment,” as it applies to a human or veterinarysubject, refers to therapeutic treatment, as well as diagnosticapplications. “Treatment” as it applies to a human or veterinarysubject, encompasses contact of the antibodies or antigen bindingfragments of the present invention to a human or animal subject.

As used herein, “therapeutically effective amount” refers to a quantityof a specific substance sufficient to achieve a desired effect in asubject being treated. For instance, this may be the amount necessary toinhibit activation of ILT3 or the amount necessary for enhancedpembrolizumab responsiveness when co-administered with pembrolizumab.

As used herein the term “PD-1” refers to the programmed Death 1 (PD-1)protein, an inhibitory member of the extended CD28/CTLA-4 family of Tcell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82;Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28family include CD28, CTLA-4, ICOS and BTLA. The PD-1 gene encodes a 55kDa type I transmembrane protein (Agata et al. (1996) Int Immunol.8:765-72). Two ligands for PD-1 have been identified, PD-L1 (B7-H1) andPD-L2 (B7-DC), that have been shown to downregulate T cell activationupon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34;Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-1 is known as animmunoinhibitory protein that negatively regulates TCR signals (Ishida,Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec.29) Immunol. Immunother. 56(5):739-745). The interaction between PD-1and PD-L1 can act as an immune checkpoint, which can lead to, e.g., adecrease in tumor infiltrating lymphocytes, a decrease in T-cellreceptor mediated proliferation, and/or immune evasion by cancerouscells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005)Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin.Cancer Res. 10:5094-100). Immune suppression can be reversed byinhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effectis additive when the interaction of PD-1 with PD-L2 is blocked as well(Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al.(2003) J. Immunol. 170:1257-66).

Antibodies and Antigen Binding Fragments

The present invention provides isolated chimeric, humanized, and humanantibodies and antigen binding fragments that specifically bind ILT3 andhave no measurable binding to closely related proteins (e.g., ILT5,ILT7, ILT8, and ILT11) as determined in a cell ELISA or Biacore assayusing 10 μg/mL antibody. The anti-ILT3 antibodies increase activity ofantigen presenting cells and dendritic cells, reduce activity ofmonocyte repressors, and increase priming of T-cells. Thus, the presentinvention further includes the use of the anti-ILT3 antibodies inmonotherapies for the treatment of cancers and for use in combinationwith anti-PD-1 or anti-PD-L1 antibodies, for either in a first line,second line, or third line therapy for the treatment of cancer.

An anti-ILT3 antibody includes any antibody disclosed herein by aminoacid sequence and includes any antibody that comprises (i) at least one,two, three, four, five, or six CDRs of an antibody disclosed herein byamino acid sequence or (ii) has no CDR amino acid sequence disclosedherein but which binds the same epitope on ILT3 as an antibody disclosedherein by amino acid sequence and which may modulate ILT3 receptorsignaling such that the antibody increases activity of antigenpresenting cells and dendritic cells, reduces activity of monocyterepressors, and increases priming of T-cells. In particular aspects, theantibody has no measurable binding to human ILT5, human ILT7, humanILT8, and human ILT11 as determined in a cell ELISA or in a Biacoreassay using 10 μg/mL of the antibody. The term specifically excludesantibodies comprising at least one CDR of antibody ZM4.1 or antibody9B11 or any of the other antibodies disclosed in U.S. Pat. Nos.7,777,008 and 8,901,281 or in U.S. Pub. Nos. 20090202544, 20150110714,20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569,WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300,and WO2018148494.

An anti-ILT3 antigen binding fragment and the like includes any proteinor peptide containing molecule that comprises (i) at least a portion ofan anti-ILT3 antibody disclosed herein by amino acid sequence, (ii) atleast one, two, three, four, five, or six CDRs of an antibody disclosedherein by sequence, or (iii) has no CDR amino acid sequence disclosedherein but which binds the same epitope on ILT3 as an anti-ILT3 antibodydisclosed herein by amino acid sequence, and which may modulate ILT3receptor signaling such that the antigen binding fragment increasesactivity of antigen presenting cells and dendritic cells, reducesactivity of monocyte repressors, and increases priming of T-cells. Inparticular aspects, the antigen binding fragment has no measurablebinding to human ILT5, human ILT7, human ILT8, and human ILT11 asdetermined in a cell ELISA or in a Biacore assay using 10 μg/mL of theanti-ILT3 antigen binding fragment. The term specifically excludesantigen binding fragments comprising at least one CDR of antibody ZM4.1or antibody 9B11 or any of the other antibodies disclosed in U.S. Pat.Nos. 7,777,008 and 8,901,281 or U.S. Pub. Nos. 20090202544, 20150110714,20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569,WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300,and WO2018148494.

In a further embodiment, an anti-ILT3 antibody includes any antibodythat comprises (i) at least HC-CDR3 of an antibody disclosed herein byamino acid sequence or (ii) has no H3-CDR3 amino acid sequence disclosedherein but which binds the same epitope on ILT3 as an antibody disclosedherein by amino acid sequence and which may modulate ILT3 receptorsignaling such that the antibody increases activity of antigenpresenting cells and dendritic cells, reduces activity of monocyterepressors, and increases priming of T-cells. In particular aspects, theantibody has no measurable binding to human ILT5, human ILT7, humanILT8, and human ILT11 as determined in a cell ELISA or in a Biacoreassay using 10 μg/mL of the antibody. The term specifically excludesantibodies comprising at least one CDR of antibody ZM4.1 or antibody9B11 or any of the other antibodies disclosed in U.S. Pat. Nos.7,777,008 and 8,901,281 or in U.S. Pub. Nos. 20090202544, 20150110714,20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569,WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300,and WO2018148494.

An anti-ILT3 antigen binding fragment and the like includes any proteinor peptide containing molecule that comprises (i) at least a portion ofan anti-ILT3 antibody disclosed herein by amino acid sequence, (ii) atleast the HC-CDR3 of an antibody disclosed herein by sequence, or (iii)has no HC-CDR3 amino acid sequence disclosed herein but which binds thesame epitope on ILT3 as an anti-ILT3 antibody disclosed herein by aminoacid sequence, and which may modulate ILT3 receptor signaling such thatthe antigen binding fragment increases activity of antigen presentingcells and dendritic cells, reduces activity of monocyte repressors, andincreases priming of T-cells. In particular aspects, the antigen bindingfragment has no measurable binding to human ILT5, human ILT7, humanILT8, and human ILT11 as determined in a cell ELISA or in a Biacoreassay using 10 μg/mL of the anti-ILT3 antigen binding fragment. The termspecifically excludes antigen binding fragments comprising at least oneCDR of antibody ZM4.1 or antibody 9B11 or any of the other antibodiesdisclosed in U.S. Pat. Nos. 7,777,008 and 8,901,281 or U.S. Pub. Nos.20090202544, 20150110714, 20150139986, and 20170267759; and, Intl. Pub.Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641,WO2016127427, WO2018089300, and WO2018148494.

In particular embodiments, the anti-ILT3 antibody is a human orhumanized anti-ILT3 antibody or antigen binding fragment or a chimericanti-ILT3 antibody or antigen binding fragment that comprises HC-CDR3 ofan anti-ILT3 antibody molecule disclosed herein or an H3-CDR3 shown inTable 3.

In particular embodiments, the anti-ILT3 antibody is a human orhumanized anti-ILT3 antibody or antigen binding fragment or a chimericanti-ILT3 antibody or antigen binding fragment that comprises HC-CDR1,HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 of an anti-ILT3 antibodymolecule disclosed herein or in Table 3.

TABLE 3 Seq Seq Seq mAb HC-CDR1 No. HC-CDR2 No. HC-CDR3 No. 52B8 NYGMS17 TISGGGDYTMYPDSVRG 20 RLWFRSLYYAMDY 23 40A6 SYSIN 47 RFWYDEGIAYNLTLES48 DRDTVGITGWFAY 49 16B1 NYCVN 55 RFWFDEGKAYNLTLES 56 DRDTVGITGWFAY 5711D1 TYWIE 63 EILPGNGNTHFNENFKD 64 RRLGRGPFDF 65 17H12 NFDMA 71SITYDGGSTSYRDSVKG 72 VESIATISTYFDY 73 37C8 SYCVN 79 RFWYDEGKVYNLTLES 80DRDTMGITGWFAY 81 1G12 TYWIQ 87 EILPGSGTTNYNENFKG 88 RLGRGPFDY 89 20E4SYSVN 95 RFWYDGGTAYNSTLES 96 DRDTMGITGWFAY 97 24A4 SYCVN 103RFWYDEGKVYNLTLES 104 DRDTLGITGWFAY 105 mAb LC-CDR1 LC-CDR2 LC-CDR3 52B8RASEKVDSFGQSFMH 41 LTSNLDS 43 QQNNEDPYT 44 40A6 KASQSVGVNVD 50 GSANRHT51 LQYGSVPYT 52 16B1 KASQSVGINVD 58 GSANRHT 59 LQYGSVPYT 60 11D1KASQDINEYIG 66 YTSTLQS 67 LQYANPLPT 68 17H12 RASQSVSMSRYDLIH 74 RASDLAS75 QQTRKSPPT 76 37C8 KASQSVGINVD 82 GSANRHT 83 LQYGSVPYT 84 1G12EASQDINKHID 90 YASILQP 91 LQYDNLLPT 92 20E4 KASQSVGVNVD 98 GSANRHT 99LQYGSVPYT 100 24A4 KASQSVGINVD 106 GSANRHT 107 LQYGSVPYT 108

In particular embodiments, the anti-ILT3 antibody is a human orhumanized anti-ILT3 antibody or antigen binding fragment or a chimericanti-ILT3 antibody or antigen binding fragment, in each case comprisinga heavy chain variable domain (V_(H)) having a heavy chaincomplementarity determining region (HC-CDR) 3 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 22, 49, 57,65, 73, 81, 89, 97, and 105, or an amino acid sequence that has 3, 2, or1 differences with an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105. In afurther embodiment, the antibody or antigen binding fragment binds to anepitope on the human ILT3, wherein the epitope comprises at least oneamino acid from one or more of the amino acid sequences set forth in inthe group consisting of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In a furtherembodiment, the antibody or antigen binding fragment binds to an epitopeon the human ILT3, wherein the epitope comprises the amino acidsequences set forth in in the group consisting of SEQ ID NO: 3, 4, 5, 6,7, and 8. In particular embodiments the amino acid sequence differencesare conservative changes/substitutions.

In particular embodiments, the anti-ILT3 antibody is a humanized orchimeric anti-ILT3 antibody disclosed herein. In particular embodiments,the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody orantigen binding fragment or a chimeric anti-ILT3 antibody or antigenbinding fragment that binds the same epitope bound by an anti-ILT3antibody disclosed herein or competes with the binding of an anti-ILT3antibody disclosed herein and the antibody comprises less than three ornone of the CDRs of an anti-ILT3 antibody disclosed herein.

The present invention further provides an antibody or antigen bindingfragment comprising (i) at least the six complementary determiningregions (CDRs) of an anti-immunoglobulin-like transcript 3 (ILT3)antibody or (ii) at least the six CDRs of an anti-ILT3 antibody whereinone or more of the six CDRs has one, two, or three amino acidsubstitutions, additions, deletions, or combinations; wherein the sixCDRs of the anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 havingthe amino acid sequence set forth in SEQ ID NO: 17, 47, 55, 63, 71, 79,87, 95, or 103; an HC-CDR2 having the amino acid sequence set forth inSEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; an HC-CDR3 having theamino acid sequence set forth in SEQ ID NO: 22, 49, 57, 65, 73, 81, 89,97, or 105; a light chain (LC)-CDR1 having the amino acid sequence setforth in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98, or 106; an LC-CDR2having the amino acid sequence set forth in SEQ ID NO: 43, 51, 59, 67,75, 83, 91, 99, or 107; and an LC-CDR3 having the amino acid sequenceset forth in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100, or 108; and,wherein the antibody or antigen binding fragment specifically bindshuman or rhesus ILT3 or both human and rhesus ILT3. In particularembodiments the amino acid sequence differences are conservativechanges/substitutions.

In particular embodiments, the present invention provides an antibody orantigen binding fragment comprising the six CDRs of the anti-ILT3antibody comprise a heavy chain (HC)-CDR1 having the amino acid sequenceset forth in SEQ ID NO:17; an HC-CDR2 having the amino acid sequence setforth in SEQ ID NO:19, 20, or 21; an HC-CDR3 having the amino acidsequence set forth in SEQ ID NO: 23, 24, 25, or 26; a light chain(LC)-CDR1 having the amino acid sequence set forth in SEQ ID NO: 34, 35,36, 37, 38, 39, 40, 41, or 42; an LC-CDR2 having the amino acid sequenceset forth in SEQ ID NO: 43; and an LC-CDR3 having the amino acidsequence set forth in SEQ ID NO: 44.

In particular embodiments, the present invention provides an antibody orantigen binding fragment comprising the six CDRs of the anti-ILT3antibody having a heavy chain (HC)-CDR1 having the amino acid sequenceset forth in SEQ ID NO: 17; an HC-CDR2 having the amino acid sequenceset forth in SEQ ID NO: 20; an HC-CDR3 having the amino acid sequenceset forth in SEQ ID NO: 23; a light chain (LC)-CDR1 having the aminoacid sequence set forth in SEQ ID NO: 41; an LC-CDR2 having the aminoacid sequence set forth in SEQ ID NO: 43; and an LC-CDR3 having theamino acid sequence set forth in SEQ ID NO: 44.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody or antigenbinding fragment comprises a heavy chain variable domain (V_(H)) havinga framework selected from the human V_(H)1, V_(H)2, V_(H)3, V_(H)4,V_(H)5, and V_(H)6 family and variants thereof having 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof; and, (b) a light chain variable domain (V_(L))having a framework selected from the human V_(κ)1, V_(κ)2, V_(κ)3,V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6,V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10 family and variants thereof having1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody comprises ahuman IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constant domain orvariant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof compared tothe amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody comprises ahuman kappa or lambda light chain constant domain or variant thereofcomprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof compared to the amino acidsequence of the native human kappa or lambda light chain domain.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody comprises (i)a human heavy chain variable domain (V_(H)) having a framework selectedfrom the human V_(H)3 family and a human light chain variable domain(V_(L)) having a framework selected from the human V_(κ)1, V_(κ)3, andV_(κ)4 family; (ii) a human IgG1 or IgG4 heavy chain (HC) constantdomain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions, additions, deletions, or combinations thereofcompared to the amino acid sequence of the native IgG1 or IgG4 isotype;and, (iii) a human kappa or lambda light chain constant domain orvariant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof compared tothe amino acid sequence of the native human kappa or lambda light chaindomain. In particular embodiments the amino acid sequence differencesare conservative changes/substitutions.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody or antigenbinding fragment comprises a heavy chain variable domain (V_(H)) and alight chain variable domain (V_(L)) having the amino acid sequences setforth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; SEQ ID NO: 45and SEQ ID NO: 46, respectively; SEQ ID NO: 53 and SEQ ID NO: 54,respectively; SEQ ID NO:61 and SEQ ID NO: 62, respectively; SEQ ID NO:69 and SEQ ID NO: 70, respectively; SEQ ID NO:77 and SEQ ID NO: 78,respectively; SEQ ID NO: 85 and SEQ ID NO: 86, respectively; SEQ ID NO:93 and SEQ ID NO: 94, respectively; or SEQ ID NO: 101 and SEQ ID NO:102, respectively.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody or antigenbinding fragment comprises a heavy chain variable domain (V_(H)) havingthe amino acid sequence set forth in SEQ ID NO: 117, 118, 119, 120, 121,122, 123, 124, or 125 and a light chain variable domain (V_(L)) havingthe amino acid sequence set forth in SEQ ID NO: 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody or antigenbinding fragment comprises a heavy chain variable domain (V_(H)) havingthe amino acid sequence set forth in SEQ ID NO: 118 and a light chainvariable domain (V_(L)) having the amino acid sequence set forth in SEQID NO: 140.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein, the antibody comprises aheavy chain (HC) constant domain comprising the amino acid sequence setforth in SEQ ID NO: 9, 10, 11, 12, or 13 and variants of SEQ ID NO: 9,11, 12, or 13 in which the HC lacks a C-terminal Lysine orglycine-lysine.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein, the antibody comprises alight chain (LC) constant domain comprising the amino acid sequence setforth in SEQ ID NO: 14.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein, the antibody comprises aheavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 142,143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178,182, 183, 184, 185, 186, 187, 191, 192, or 193 and variants of an HCcomprising the amino acid sequence of SEQ ID NO: 143, 144, 148, 149,150, 167, 168, 169, 170, 174, or 175 in which the HC lacks a C-terminalLysine or glycine-lysine.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein, the antibody comprises alight chain (LC) comprising the amino acid sequence set forth in SEQ IDNO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, or 166.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein, the antibody comprises aheavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 142,143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178,182, 183, 184, 185, 186, 187, 191, 192, or 193 and a light chain (LC)comprising the amino acid sequence set forth in SEQ ID NO: 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166,and variants of an HC comprising the amino acid sequence of SEQ ID NO:143, 144, 148, 149, 150, 167, 168, 169, 170, 174, or 175 in which the HClacks a C-terminal Lysine or glycine-lysine.

In particular embodiments, the present invention provides an antibodyselected from the antibodies presented in Table 4.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein, the antibody comprises aheavy chain (HC) having the amino acid sequence set forth in SEQ ID NO:143 and a light chain (LC) comprising the amino acid sequence set forthin SEQ ID NO: 165 and variants in which the HC lacks a C-terminal Lysineor glycine-lysine.

In particular embodiments, the present invention provides the aboveantibody or antigen binding fragment wherein the antibody comprises ahuman IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constant domain orvariant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof compared tothe amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype,and variants thereof in which the HC lacks a C-terminal Lysine orglycine-lysine.

In some embodiments, different constant domains may be fused to a V_(L)and V_(H) regions comprising the CDRs provided herein. In particularembodiments, the V_(H) regions comprising the CDRs provided herein maybe fused to a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constantdomain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions, additions, deletions, or combinations thereofcompared to the amino acid sequence of the native or wild-type IgG1,IgG2, IgG3, or IgG4 isotype, and variants thereof in which the HC lacksa C-terminal Lysine or glycine-lysine.

In particular embodiments, the anti-ILT3 antibody (or antigen bindingfragment) has an altered effector function and may comprise a heavychain constant domain other than native (wild-type) human IgG1, forexample a human IgG1 that has mutations that abrogate or minimize one ormore effector functions, including ability to bind complement, humanIgG4, or a hybrid human IgG1/human IgG4, and variants thereof in whichthe HC lacks a C-terminal Lysine or glycine-lysine.

Although native human IgG1 antibodies provide for long half-life and foreffector functions, such as complement activation and antibody-dependentcellular cytotoxicity, such activities may not be desirable for all usesof an antibody. Thus, in particular embodiments, it is desirable thatthe heavy chain constant domain or Fc have minimal or reduced effectorfunction (“effector-less”). In those instances, the anti-ILT3 HCvariable domain may be fused to a human IgG4 constant domain, which isgenerally known to be effector-less, or an IgG1 constant domain that hasbeen mutated to be rendered effecter-less. These effector-less moleculeshave minimal or reduced binding to human FcγRIIIA, and FcγRIIA, andFcγ.RI compared to the polypeptide comprising the wildtype IgG Fcregion, wherein the affinity to each of human FcγRIIIA, and FcγRIIA, andFcγRI is reduced by 1.15-fold to 100-fold compared to the polypeptidecomprising the wildtype IgG constant domain, and wherein theantibody-dependent cell-mediated cytotoxicity (ADCC) induced by saidmolecule is 0-20% of the ADCC induced by the polypeptide comprising thewild-type human IgG1 constant domain.

Therefore in particular embodiments, the present invention includeschimeric or humanized anti-ILT3 antibodies and antigen-binding fragmentsthereof that comprise a human IgG4 constant domain. In a furtherembodiment, the human IgG4 constant domain may be modified to differfrom the native (wild-type) human IgG4 constant domain (Swiss-ProtAccession No. P01861.1) at a position corresponding to position 228 inthe EU system and position 241 in the Kabat system in which the nativeserine at position 108 (Ser108) of the HC constant domain is replacedwith proline (Pro), see for example SEQ ID NO: 9. This modificationprevents formation of a potential inter-chain disulfide bond between thecysteine at position 106 (Cys106) and the cysteine at position 109(Cys109), which correspond to positions Cys226 and Cys229 in the EUsystem and positions Cys239 and Cys242 in the Kabat system, which mayinterfere with proper intra-chain disulfide bond formation. See Angal etal. Mol. Imunol. 30:105 (1993); see also (Schuurman et. al., Mol.Immunol. 38: 1-8, (2001); SEQ ID NOs: 14 and 41). In particularembodiments, the human IgG4 constant domain may further include inaddition to the S228P substitution an L235E substitution.

In another embodiment, the chimeric or humanized anti-ILT3 antibody maybe fused to a modified human IgG1 constant domain, which has beenmodified to be effector-less. In one embodiment, the human IgG1 HC mayinclude substitutions of human IgG2 HC residues at positions 233-236 andIgG4 HC residues at positions 327, 330, and 331 to greatly reduce ADCCand CDC (Armour et al., Eur J Immunol. 29(8):2613-24 (1999); Shields etal., J Biol Chem. 276(9):6591-604(2001)). In particular embodiments, theantibody comprises a human IgG1 heavy chain (HC) constant domain orvariant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof compared tothe amino acid sequence of the native IgG, which provides an antibodyhaving reduced or minimal effector function. In particular aspects, theIgG1 has been modified to comprise or consist of an L234A, an L235A, anda D265S mutation to render the Fc effector-less. Other mutations thatmay be used to render an IgG1 Fc effector-less may be found in U.S. Pat.No. 8,969,526.

In another embodiment, the human IgG1 HC is modified to lackN-glycosylation of the asparagine (Asn) residue at around position 297of the HC. The consensus sequence for N-glycosylation is Asn-Xaa-Ser/Thr(wherein Xaa is any amino acid except Pro); in IgG1 the N-glycosylationconsensus sequence is Asn-Ser-Thr. The modification may be achieved byreplacing the codon for the Asn at position 297 in the nucleic acidmolecule encoding the HC with a codon for another amino acid, forexample Gln. Alternatively, the codon for Ser may be replaced with thecodon for Pro or the codon for Thr may be replaced with any codon exceptthe codon for Ser, e.g. N297A or N297D. Such modified IgG1 moleculeshave little or no detectable effector function. Alternatively, all threecodons are modified.

In another embodiment, the human IgG1 constant domain is modified toinclude one or more amino acid substitutions selected from E233P, L234A,L235A, L235E, N297A, N297D, D265S, and P331S, wherein the residues arenumbered according to the EU index of Kabat, and wherein saidpolypeptide exhibits a reduced affinity to the human FcγRIIIA and/orFcγRIIA and/or FcγRI compared to a polypeptide comprising the wildtypeIgG constant domain region. In particular embodiments, the human IgGconstant domain comprises substitutions of L234A, L235A, and D265S asillustrated by SEQ ID NO: 4, for example. In particular embodiments, thehuman IgG1 constant domain comprises an amino acid substitution atposition Pro329 and at least one further amino acid substitution E233P,L234A, L235A, L235E, N297A, N297D, D265S, and P331. These and othersubstitutions are disclosed in WO9428027; WO2004099249; WO20121300831,U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature406, 267-273 (20 Jul. 2000)).

In an embodiment of the invention, the anti-ILT3 antibodies or antigenbinding fragments thereof include embodiments in which one or more ofthe six CDRs has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof comprise a full tetrameric structurehaving two light chains and two heavy chains, including constantregions. The variable regions of each light chain/heavy chain pair formthe antibody binding site. Thus, in general, an intact antibody has twobinding sites. Except in bispecific antibodies, the two binding sitesare, in general, the same.

In specific embodiments, the present invention provides the anti-ILT3antibodies shown in the Table 4. With the exception of those antibodiescomprising a replacement of the tryptophan residue at position 101 ofthe V_(H), the antibodies disclosed herein bind the human ILT3.

TABLE 4 SEQ ID NO: mAb Heavy Light No. Description Chain Chain  1Humanized anti-ILT3 mAb (52B8 VH1/VL1) IgG4 142 151 S228P/Kappa  2Humanized anti-ILT3 mAb (52B8 VH1/VL2) IgG4 142 152 S228P/Kappa  3Humanized anti-ILT3 mAb (52B8 VH1/VL3) IgG4 142 153 S228P/Kappa  4Humanized anti-ILT3 mAb (52B8 VH1/VL4) IgG4 142 154 S228P/Kappa  5Humanized anti-ILT3 mAb (52B8 VH2/VL1) IgG4 148 151 S228P/Kappa  6Humanized anti-ILT3 mAb (52B8 VH2/VL2) IgG4 148 152 S228P/Kappa  7Humanized anti-ILT3 mAb (52B8 VH2/VL3) IgG4 148 153 S228P/Kappa  8Humanized anti-ILT3 mAb (52B8 VH2/VL4) IgG4 148 154 S228P/Kappa  9Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL1) 143 151 IgG4 S228P/Kappa 10Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2) 143 152 IgG4 S228P/Kappa 11Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL3) 143 153 IgG4 S228P/Kappa 12Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL4) 143 154 IgG4 S228P/Kappa 13Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL1) 149 151 IgG4 S228P/Kappa 14Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL2) 149 152 IgG4 S228P/Kappa 15Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL3) 149 153 IgG4 S228P/Kappa 16Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL4) 149 154 IgG4 S228P/Kappa 17Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL1) 144 151 IgG4 S228P/Kappa 18Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL2) 144 152 IgG4 S228P/Kappa 19Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL3) 144 153 IgG4 S228P/Kappa 20Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL4) 144 155 IgG4 S228P/Kappa 21Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL1) 150 151 IgG4 S228P/Kappa 22Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL2) 150 152 IgG4 S228P/Kappa 23Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL3) 150 153 IgG4 S228P/Kappa 24Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL4) 150 154 IgG4 S228P/Kappa 25Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL2) 169 152 L234A L235A D265S)IgG1/Kappa 26 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL5) 169 155 L234AL235A D265S) IgG1/Kappa 27 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL6)169 156 L234A L235A D265S) IgG1/Kappa 28 Humanized anti-ILT3 mAb ((52B8VH1 M64V/VL7) 169 157 L234A L235A D265S) IgG1/Kappa 29 Humanizedanti-ILT3 mAb ((52B8 VH1 M64V/VL8) 169 158 L234A L235A D265S) IgG1/Kappa30 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5) 143 155 IgG4 S228P/Kappa31 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL6) 143 156 IgG4 S228P/Kappa32 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL7) 143 157 IgG4 S228P/Kappa33 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL8) 143 158 IgG4 S228P/Kappa34 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 152 VL2) IgG4S228P/Kappa 35 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 152VL2) IgG4 S228P/Kappa 36 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/147 152 VL2) IgG4 S228P/Kappa 37 Humanized anti-ILT3 mAb ((52B8 VH1 M64VW101F/ 145 152 VL2) L234A L235A D265S) IgG1/Kappa 38 Humanized anti-ILT3mAb ((52B8 VH1 M64V W101Y/ 146 152 VL2) L234A L235A D265S) IgG1/Kappa 39Humanized anti-ILT3 mAb ((52B8 VH1 M64V W101Q/ 147 152 VL2) L234A L235AD265S) IgG1/Kappa 40 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 143 159S35A) IgG4 S228P/Kappa 41 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 143160 S35N) IgG4 S228P/Kappa 42 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2143 161 N34Q) IgG4 S228P/Kappa 43 Humanized anti-ILT3 mAb (52B8 VH1M64V/VL2 143 162 N34D) IgG4 S228P/Kappa 44 Humanized anti-ILT3 mAb (52B8VH1 M64V/VL5 143 163 S35A) IgG4 S228P/Kappa 45 Humanized anti-ILT3 mAb(52B8 VH1 M64V/VL5 143 164 S35N) IgG4 S228P/Kappa 46 Humanized anti-ILT3mAb (52B8 VH1 M64V/VL5 143 165 N34Q) IgG4 S228P/Kappa 47 Humanizedanti-ILT3 mAb (52B8 VH1 M64V/VL5 143 166 N34D) IgG4 S228P/Kappa 48Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 155 VL5) IgG4S228P/Kappa 49 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 155VL5) IgG4 S228P/Kappa 50 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/147 155 VL5) IgG4 S228P/Kappa 51 Humanized anti-ILT3 mAb (52B8 VH1 M64VW101F/ 145 163 VL5 S35A) IgG4 S228P/Kappa 52 Humanized anti-ILT3 mAb(52B8 VH1 M64V W101F/ 145 164 VL5 S35N) IgG4 S228P/Kappa 53 Humanizedanti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 165 VL5 N34Q) IgG4 S228P/Kappa54 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 166 VL5 N34D) IgG4S228P/Kappa 55 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 163 VL5S35A) IgG4 S228P/Kappa 56 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/146 164 VL5 S35N) IgG4 S228P/Kappa 57 Humanized anti-ILT3 mAb (52B8 VH1M64V W101Y/ 146 165 VL5 N34Q) IgG4 S228P/Kappa 58 Humanized anti-ILT3mAb (52B8 VH1 M64V W101Y/ 146 166 VL5 N34D) IgG4 S228P/Kappa 59Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 163 VL5 S35A) IgG4S228P/Kappa 60 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 164 VL5S35N) IgG4 S228P/Kappa 61 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/147 165 VL5 N34Q) IgG4 S228P/Kappa 62 Humanized anti-ILT3 mAb (52B8 VH1M64V W101Q/ 147 166 VL5 N34D) IgG4 S228P/Kappa 63 Humanized anti-ILT3mAb (52B8 VH1 M64V/VL1 210 126 N34Q) IgG1 N297A/Kappa 64 Humanizedanti-ILT3 mAb (52B8 VH1 M64V/VL2 210 127 IgG1 N297A/Kappa 65 Humanizedanti-ILT3 mAb (52B8 VH1 M64V/VL2 210 161 N34Q) IgG1 N297A/Kappa 66Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL3 210 128 N34Q) IgG1N297A/Kappa 67 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL4 210 129 N34Q)IgG1 N297A/Kappa 68 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 210 130IgG1 N297A/Kappa 69 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 210 165N34Q) IgG1 N297A/Kappa 70 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL6 210131 N34Q) IgG1 N297A/Kappa 71 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL7210 132 N34Q) IgG1 N297A/Kappa 72 Humanized anti-ILT3 mAb (52B8 VH1M64V/VL8 210 133 N34Q) IgG1 N297A/Kappa 73 Chimeric anti-ILT3 52B8 mouseVH/human IgG4 113 116 (S228P): mouse VL/human Kappa 74 Chimericanti-ILT3 52B8 mouse VH M64V/human IgG4 114 116 (S228P): mouse VL/humanKappa 75 Chimeric anti-ILT3 52B8 mouse VH M64L/human IgG4 115 116(S228P): mouse VL/human Kappa 76 Chimeric anti-ILT3 52B8 mouse VH/humanIgG1 Residues 116 (N297A): mouse VL/human Kappa 1-122 of SEQ ID NO: 113And SEQ ID NO: 211 77 Chimeric anti-ILT3 52B8 mouse VH M64V/human IgG1Residues 116 (N297A): mouse VL/human Kappa 1-122 of SEQ ID NO: 114 AndSEQ ID NO: 211 78 Chimeric anti-ILT3 52B8 mouse VH/human IgG1: mouseResidues 116 VL/human Kappa 1-122 of SEQ ID NO: 113 And SEQ ID NO: 11 79Chimeric anti-ILT3 52B8 mouse VH M64V/human Residues 116 IgG1: mouseVL/human Kappa 1-122 of SEQ ID NO: 114 And SEQ ID NO: 11 80 Chimericanti-ILT3 40A6 rat VH/human IgG4 194 195 (S228P): rat VL/human Kappa 81Chimeric anti-ILT3 16B1 rat VH/human IgG4 196 197 (S228P): rat VL/humanKappa 82 Chimeric anti-ILT3 11D1 mouse VH/human IgG4 198 199 (S228P):mouse VL/human Kappa 83 Chimeric anti-ILT3 17H12 rat VH/human IgG4 200201 (S228P): rat VL/human Kappa 84 Chimeric anti-ILT3 37C8 rat VH/humanIgG4 202 203 (S228P): rat VL/human Kappa 85 Chimeric anti-ILT3 1G12mouse VH/human IgG4 203 205 (S228P): mouse VL/human Kappa 86 Chimericanti-ILT3 20E4 rat VH/human IgG4 206 207 (S228P): rat VL/human Kappa 87Chimeric anti-ILT3 24A4 rat VH/human IgG4 208 209 (S228P): rat VL/humanKappa 88 Chimeric anti-ILT3 40A6 rat VH/human IgG1 212 195 (N297A): ratVL/human Kappa 89 Chimeric anti-ILT3 16B1 rat VH/human IgG1 213 197(N297A): rat VL/human Kappa 90 Chimeric anti-ILT3 11D1 mouse VH/humanIgG1 214 199 (N297A): mouse VL/human Kappa 91 Chimeric anti-ILT3 17H12rat VH/human IgG1 215 201 (N297A): rat VL/human Kappa 92 Chimericanti-ILT3 37C8 rat VH/human IgG1 216 203 (N297A): rat VL/human Kappa 93Chimeric anti-ILT3 1G12 mouse VH/human IgG1 217 205 (N297A): mouseVL/human Kappa 94 Chimeric anti-ILT3 20E4 rat VH/human IgG1 218 207(N297A): rat VL/human Kappa 95 Chimeric anti-ILT3 24A4 rat VH/human IgG1219 209 (N297A): rat VL/human Kappa 96 Chimeric anti-ILT3 40A6 ratVH/human IgG1 220 195 (N297A): rat VL/human Kappa

Epitope mapping by hydrogen-deuterium exchange mass spectrometry(HDX-MS) as described in Example 4 shows that the anti-ILT3 antibodiesdisclosed herein bind to an epitope on the extracellular domain near theborder between the D1 and D2 domains of the extracellular domain ofILT3. The epitope identified using HDX-MS indicates that the epitopebound by the anti-ILT3 antibodies disclosed herein comprises or consistsof at least one amino acid within one or more of the peptide domainamino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In a further embodiment, the epitope comprises orconsists of one or more of the peptide domain amino acid sequencesselected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8.In certain embodiments, the epitope comprises or consists of at leastone amino acid in each of the peptide domain amino acid sequencesselected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8and identified in the HDX-MS. In particular embodiments, the epitopecomprises or consists of one or more of the peptide domain amino acidsequences selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6,7, and 8. In particular embodiments, the epitope comprises or consistsof the peptide domains shown in SEQ ID Nos: 3, 4, 5, 6, 7, and 8.

Thus, the present invention further provides a chimeric, humanized, orhuman antibody or antigen binding fragment that binds to an epitope onILT3 wherein the epitope comprises or consists of at least one aminoacid within one or more of the peptide domains comprising amino acidsequences shown by the amino acid sequences set forth in SEQ ID NOs: 3,4, 5, 6, 7, and 8 as determined by hydrogen deuterium exchange massspectrometry (HDX-MS) analysis.

In a further embodiment, the present invention further provides achimeric, humanized, or human antibody or antigen binding fragment thatbinds to an epitope on ILT3 wherein the epitope comprises or consists ofamino acids within the peptide domains shown in one or more of SEQ IDNos: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprisesor consists of at least one amino acid in each of the peptide domainsidentified in the heat map determined by HDX-MS and shown in FIG. 3A.

The present invention further provides a chimeric, humanized, or humanantibody or antigen binding fragment that cross-blocks the binding of anantibody comprising a heavy chain variable domain having the amino acidsequence set forth in SEQ ID NO: 15 and a light chain variable domainhaving the amino acid sequence shown in SEQ ID NO: 16 to an epitope onILT3. In a further embodiment, the epitope comprises or consists of atleast one amino acid within one or more of the peptide domainscomprising or consisting of amino acid sequences shown by the amino acidsequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 as determined byhydrogen deuterium exchange mass spectrometry (HDX-MS) analysis. In afurther embodiment, the epitope comprises or consists of amino acidswithin the peptide domains shown in one or more of SEQ ID NOs: 3, 4, 5,6, 7, and 8. In certain embodiments, the epitope comprises or consistsof at least one amino acid in each of the peptide domains identified inthe HDX-MS.

The present invention further provides bispecific antibodies andantigen-binding fragments comprising a first antibody or antigen bindingfragment that binds ILT3 and a second antibody or antigen bindingfragment that binds a molecule other than ILT3, wherein the firstantibody or antigen binding fragment comprises at least the amino acidsequence of an HC-CDR3 having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105,or having an amino acid sequence that has 3, 2, or 1 differences with anamino acid sequence selected from the group consisting of SEQ ID NO: 22,49, 57, 65, 73, 81, 89, 97, and 105 and wherein the first antibody bindsan ILT3 epitope comprising amino acids within the sequences of SEQ IDNos: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule otherthan ILT3, and methods of use thereof.

The present invention further provides bispecific antibodies andantigen-binding fragments comprising a first antibody or antigen bindingfragment that binds ILT3 and a second antibody or antigen bindingfragment that binds a molecule other than ILT3, wherein the firstantibody or antigen binding fragment comprising at least the six CDRs ofan anti-ILT3 antibody or embodiments thereof wherein one or more of theCDRs has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof and wherein the first antibody bindsan ILT3 epitope comprising amino acids within the sequences of SEQ IDNOs: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule otherthan ILT3, and methods of use thereof.

The present invention further provides biparatopic antibodies(antibodies having binding specificity for different epitopes on thesame antigen) having a first heavy/light chain pair of a first antibodythat comprises at least an HC-CDR3 having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73,81, 89, 97, and 105, or having an amino acid sequence that has 3, 2, or1 differences with an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105,wherein the first heavy/light chain pair binds an ILT3 epitopecomprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6,7, and 8 and the second antibody binds a molecule other than ILT3 and asecond heavy/light chain pair of a second antibody having specificityfor an anti-ILT3 epitope that is different from the epitope recognizedby the first heavy/light chain pair.

The present invention further provides biparatopic antibodies(antibodies having binding specificity for different epitopes on thesame antigen) having first heavy/light chain pair of a first antibodythat comprises at least the six CDRs of an anti-ILT3 antibody orembodiments thereof wherein one or more of the CDRs has one, two, orthree amino acid substitutions, additions, deletions, or combinationsthereof wherein the first antibody binds an ILT3 epitope comprisingamino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8,wherein the first heavy/light chain pair binds an ILT3 epitopecomprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6,7, and 8 and the second antibody binds a molecule other than ILT3 and asecond heavy/light chain pair of a second antibody having specificityfor an anti-ILT3 epitope that is different from the epitope recognizedby the first heavy/light chain pair.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-ILT3antibodies or antigen binding fragments thereof, the antibody or antigenbinding fragments thereof is admixed with a pharmaceutically acceptablecarrier or excipient. See, e.g., Remington's Pharmaceutical Sciences andU.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton,Pa. (1984) and continuously updated on the Internet by the U.S.Pharmacopeial Convention (USP) 12601 Twinbrook Parkway, Rockville, Md.20852-1790, USA.

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

In a further embodiment, a composition comprising an antibody orantibody fragment disclosed herein is administered to a subject inaccordance with the Physicians' Desk Reference 2017 (Thomson Healthcare;75st edition (Nov. 1, 2002)). Methods of administering antibodymolecules are known in the art and are described below. Suitable dosagesof the molecules used will depend on the age and weight of the subjectand the particular drug used. Dosages and therapeutic regimens of theanti-ILT3 antibody or antigen binding fragment can be determined by askilled artisan. In certain embodiments, the anti-ILT3 antibody orantigen binding fragment is administered by injection (e.g.,subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g.,about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about3 mg/kg. In some embodiments, the anti-ILT3 antibody or antigen bindingfragment is administered at a dose of about 1 mg/kg, about 3 mg/kg, or10 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 40 mg/kg. In someembodiments, the anti-ILT3 antibody or antigen binding fragment isadministered at a dose of about 1-3 mg/kg, or about 3-10 mg/kg. In someembodiments, the anti-ILT3 antibody or antigen binding fragment isadministered at a dose of about 0.5-2, 2-4, 2-5, 5-15, or 5-20 mg/kg.The dosing schedule can vary from e.g., once a week to once every 2, 3,or 4 weeks. In one embodiment, the anti-ILT3 antibody or antigen bindingfragment is administered at a dose from about 10 to 20 mg/kg every otherweek.

The mode of administration can vary. Suitable routes of administrationis preferably parenteral or subcutaneous. Other routes of administrationmay include oral, transmucosal, intradermal, direct intraventricular,intravenous, intranasal, inhalation, insufflation, or intra-arterial.

In particular embodiments, the anti-ILT3 antibodies or antigen bindingfragments thereof can be administered by an invasive route such as byinjection. In further embodiments of the invention, the anti-ILT3antibodies or antigen binding fragments thereof, or pharmaceuticalcomposition thereof, may be administered intravenously, subcutaneously,intraarterially, or by inhalation, aerosol delivery. Administration bynon-invasive routes (e.g., orally; for example, in a pill, capsule ortablet) is also within the scope of the present invention.

Compositions can be administered with medical devices known in the art.For example, a pharmaceutical composition of the invention can beadministered by injection with a hypodermic needle, including, e.g., aprefilled syringe or autoinjector.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. No. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

The pharmaceutical compositions disclosed herein may also beadministered by infusion. Examples of well-known implants and modulesform administering pharmaceutical compositions include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,447,233,which discloses a medication infusion pump for delivering medication ata precise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments. Many other suchimplants, delivery systems, and modules are well known to those skilledin the art.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody, the level ofsymptoms, the immunogenicity of the therapeutic antibody, and theaccessibility of the target cells in the biological matrix. Preferably,the administration regimen delivers sufficient therapeutic antibody toeffect improvement in the target disease state, while simultaneouslyminimizing undesired side effects. Accordingly, the amount of biologicdelivered depends in part on the particular therapeutic antibody and theseverity of the condition being treated. Guidance in selectingappropriate doses of therapeutic antibodies is available (see, e.g.,Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokinesand Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)(1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases,Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New Engl. J. Med.348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973;Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al.(2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J.Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the antibody or antibodybinding fragment and the particular therapeutic effect to be achieved,and (b) the limitations inherent in the art of compounding such anactive molecules for the treatment of sensitivity in individuals. (see,e.g., Yang, et al. (2003) New Engl. J Med. 349:427-434; Herold, et al.(2002) New Engl. J Med. 346:1692-1698; Liu, et al. (1999) J Neurol.Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol.Immunother. 52:133-144).

Use of the Anti-ILT3 Antibodies or Antigen Binding Fragments DisclosedHerein

The anti-ILT3 antibodies and antigen binding fragments disclosed hereinbeing non-promiscuous for related ILTs may be used to specificallydetect human ILT3 (e.g., in a biological sample, such as serum orplasma), using a conventional immunoassay, such as an enzyme linkedimmunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissueimmunohistochemistry. The invention thus provides a method for detectinghuman ILT3 in a biological sample comprising contacting a biologicalsample with an anti-ILT3 antibody or antigen binding fragment anddetecting either the anti-ILT3 antibody or antigen binding fragmentbound to human ILT3 or unbound anti-ILT3 antibody or antigen bindingfragment disclosed herein, to thereby detect human ILT3 in thebiological sample. The anti-ILT3 antibody or antigen binding fragment isdirectly or indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound anti-ILT3 antibody or antigen bindingfragment disclosed herein. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S, and ³H.

Alternative to labeling the anti-ILT3 antibody or antigen bindingfragment, human ILT3 can be assayed in biological fluids by acompetition immunoassay utilizing ILT3 standards labeled with adetectable substance and an unlabeled anti-human ILT3 anti-ILT3 antibodyor antigen binding fragment disclosed herein. In this assay, thebiological sample, the labeled ILT3 standards and the anti-ILT3 antibodyor antigen binding fragment are combined and the amount of labeled ILT3standard bound to the unlabeled anti-ILT3 antibody or antigen bindingfragment disclosed herein is determined. The amount of human ILT3 in thebiological sample is inversely proportional to the amount of labeledILT3 standard bound to the anti-ILT3 antibody or antigen bindingfragment.

An anti-ILT3 antibody or antigen binding fragment disclosed herein mayalso be used to detect ILT3 from a species other than humans, inparticular ILT3 from primates (e.g., cynomolgus monkey or rhesusmonkey).

Methods of Upmodulating Immune Responses In Vivo

The anti-ILT3 antibodies or antigen binding fragments disclosed hereinmay be used as immunostimulatory compositions, e.g., alone or as part ofa vaccine or combination therapy, to promote B cell, and/or T cellactivation, e.g., either Th1 or Th2 cell activation, in a subject. Thatis, the anti-ILT3 antibody or antigen binding fragment disclosed hereinmay serve as adjuvants used in combination with an antigen of interestto enhance an immune response to that antigen of interest in vivo. Forexample, to stimulate an antibody or cellular immune response to anantigen of interest (e.g., for vaccination purposes), the antigen andanti-ILT3 antibody or antigen binding fragment disclosed herein may beco-administered (e.g., co-administered at the same time in the same orseparate compositions, or sequentially in time such that an enhancedimmune response occurs). The antigen of interest and the anti-ILT3antibody or antigen binding fragment disclosed herein may be formulatedtogether into a single pharmaceutical composition or in separatecompositions. In one embodiment, the antigen of interest and theanti-ILT3 antibody or antigen binding fragment disclosed herein areadministered simultaneously to the subject. Alternatively, in certainsituations it may be desirable to administer the antigen first and thenthe anti-ILT3 antibody or antigen binding fragment disclosed herein orvice versa (for example, in the case of an antigen that naturally evokesa Th1 response, it may be beneficial to first administer the antigenalone to stimulate a Th1 response and then administer an anti-ILT3antibody or antigen binding fragment disclosed herein, alone or togetherwith a boost of antigen, to shift the immune response to a Th2response). In preferred embodiments, an anti-ILT3 antibody or antigenbinding fragment disclosed herein is administered at the time of primingwith antigen, i.e., at the time of the first administration of antigen.For example, day −3, −2, −1, 0, +1, +2, +3. A particularly preferred dayof administration of an anti-ILT3 antibody or antigen binding fragmentdisclosed herein is day −1.

In one embodiment, an anti-ILT3 antibody or antigen binding fragmentdisclosed herein is administered with an antigen of interest. An antigenof interest is one to which an immune response is desired. For example,an antigen of interest is an antigen capable of stimulating immuneprotection in a subject against challenge by an infectious agent fromwhich the antigen was derived. Further contemplated is administration ofan anti-ILT3 antibody or antigen binding fragment disclosed herein toincrease immune responses without having to administer an antigen.

Exemplary antigens of interest therefore include those derived frominfectious agents, wherein an immune response directed against theantigen serves to prevent or treat disease caused by the agent. Suchantigens include, but are not limited to, viral, bacterial, fungal orparasite proteins and any other proteins, glycoproteins, lipoprotein,glycolipids, and the like. Antigens of interest also include those whichprovide benefit to a subject which is at risk for acquiring or which isdiagnosed as having a tumor. The subject is preferably a mammal and mostpreferably, is a human.

Typical antigens of interest may be classified as follows: proteinantigens, such as ceruloplasmin and serum albumin; bacterial antigens,such as teichoic acids, flagellar antigens, capsular polysaccharides,and extra-cellular bacterial products and toxins; glycoproteins andglycolipids; viruses, such as animal, plant, and bacterial viruses;conjugated and synthetic antigens, such as protein/hapten conjugates,molecules expressed preferentially by tumors, compared to normal tissue;synthetic polypeptides; and nucleic acids, such as ribonucleic acid anddeoxyribonucleic acid. The term “infectious agent,” as used herein,includes any agent which expresses an antigen, which elicits a hostcellular immune response. Non-limiting examples of viral antigens whichmay be considered useful as include, but are not limited to, thenucleoprotein (NP) of influenza virus and the Gag proteins of HIV. Otherheterologous antigens include, but are not limited to, HIV Env proteinor its component parts gp120 and gp41, HIV Nef protein, and the HIV Polproteins, reverse transcriptase and protease. In addition, other viralantigens such as Ebola virus (EBOV) antigens, such as, for example, EBOVNP or glycoprotein (GP), either full-length or GP deleted in the mucinregion of the molecule (Yang et al., Nat Med 6:886 (2000), small poxantigens, hepatitis A, B or C virus, human rhinovirus such as type 2 ortype 14, herpes simplex virus, poliovirus type 2 or 3, foot-and-mouthdisease virus (FMDV), rabies virus, rotavirus, influenza virus,coxsackie virus, human papilloma virus (HPV), for example the type 16papilloma virus, the E7 protein thereof, and fragments containing the E7protein or its epitopes; and simian immunodeficiency virus (SIV) may beused. The antigens of interest need not be limited to antigens of viralorigin. Parasitic antigens, such as, for example, malarial antigens areincluded, as are fungal antigens, bacterial antigens and tumor antigens.Examples of antigens derived from bacteria are those derived fromBordetella pertussis (e.g., P69 protein and filamentous haemagglutinin(FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E. coliantigens such as E. coli heat labile toxin B subunit (LT-B), E. coli K88antigens, and enterotoxigenic E. coli antigens. Other examples ofantigens include Schistosoma mansoni P28 glutathione S-transferaseantigens (P28 antigens) and antigens of flukes, mycoplasma, roundworms,tapeworms, Chlamydia trachomatis, and malaria parasites, e.g., parasitesof the genus plasmodium or babesia, for example Plasmodium falciparum,and peptides encoding immunogenic epitopes from the aforementionedantigens.

By the term “tumor-related antigen,” as used herein, is meant an antigenwhich affects tumor growth or metastasis in a host organism. Thetumor-related antigen may be an antigen expressed by a tumor cell, or itmay be an antigen that is expressed by a non-tumor cell but when soexpressed, promotes the growth or metastasis of tumor cells. The typesof tumor antigens and tumor-related antigens include any known orheretofore unknown tumor antigen, including, without limitation, thebcr/abl antigen in leukemia, HPVE6 and E7 antigens of the oncogenicvirus associated with cervical cancer, the MAGE1 and MZ2-E antigens inor associated with melanoma, and the MVC-1 and HER-2 antigens in orassociated with breast cancer.

An infection, disease or disorder which may be treated or prevented bythe administration of a composition comprising an anti-ILT3 antibody orantigen binding fragment disclosed herein includes any infection,disease or disorder wherein a host immune response acts to prevent theinfection, disease or disorder. Diseases, disorders, or infection whichmay be treated or prevented by the administration of a compositioncomprising an anti-ILT3 antibody or antigen binding fragment disclosedherein include, but are not limited to, any infection, disease ordisorder caused by or related to a fungus, parasite, virus, or bacteria,diseases, disorders or infections caused by or related to various agentsused in bioterrorism, listeriosis, Ebola virus, SARS, small pox,hepatitis A, hepatitis B, hepatitis C, diseases and disorders caused byhuman rhinovirus, HIV and AIDS, Herpes, polio, foot-and-mouth disease,rabies, diseases or disorders caused by or related to: rotavirus,influenza, coxsackie virus, human papilloma virus, SIV, malaria, cancer,e.g., tumors, and diseases or disorders caused by or related toinfection by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis,E. coli, flukes, mycoplasma, roundworms, tapeworms, Chlamydiatrachomatis, and malaria parasites, etc.

Immune Responses to Tumor Cells

Regulatory T cells play an important role in the maintenance ofimmunological self-tolerance by suppressing immune responses againstautoimmune diseases and cancer. Accordingly, in one embodiment,upmodulating an immune response would be beneficial for enhancing animmune response in cancer. Therefore, the anti-ILT3 antibodies orantigen binding fragments disclosed herein may be used in the treatmentof malignancies, to inhibit tumor growth or metastasis. The anti-ILT3antibodies or antigen binding fragments disclosed herein may beadministered systemically or locally to the tumor site.

In one embodiment, modulation of human ILT3 function may be useful inthe induction of tumor immunity. An ILT3 binding molecule may beadministered to a patient having tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) to overcome tumor-specifictolerance in the subject.

As used herein, the term “neoplastic disease” is characterized bymalignant tumor growth or in disease states characterized by benignhyperproliferative and hyperplastic cells. The common medical meaning ofthe term “neoplasia” refers to “new cell growth” that results as a lossof responsiveness to normal growth controls, e.g., neoplastic cellgrowth.

As used herein, the terms “hyperproliferative”, “hyperplastic”,malignant” and “neoplastic” are used interchangeably, and refer to thosecells in an abnormal state or condition characterized by rapidproliferation or neoplasia. The terms are meant to include all types ofhyperproliferative growth, hyperplastic growth, cancerous growths oroncogenic processes, metastatic tissues or malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. A “hyperplasia” refers to cells undergoing anabnormally high rate of growth. However, as used herein, the termsneoplasia and hyperplasia can be used interchangeably, as their contextwill reveal, referring generally to cells experiencing abnormal cellgrowth rates. Neoplasias and hyperplasias include “tumors,” which may beeither benign, premalignant or malignant.

The terms “neoplasia,” “hyperplasia,” and “tumor” are often commonlyreferred to as “cancer,” which is a general name for more than 100disease that are characterized by uncontrolled, abnormal growth ofcells. Examples of cancer include, but are not limited to: breast;colon; non-small cell lung, head and neck; colorectal; lung; prostate;ovary; renal; melanoma; and gastrointestinal (e.g., pancreatic andstomach) cancer; and osteogenic sarcoma.

In one embodiment, the cancer is selected from the group consisting of:pancreatic cancer, melanomas, breast cancer, lung cancer, head and neckcancer, bronchus cancer, colorectal cancer, prostate cancer, pancreaticcancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain orcentral nervous system cancer (e.g., gliobastoma), peripheral nervoussystem cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, and cancer of hematologicaltissues.

Immune Responses to Infectious Agents

Upregulation of immune responses may be in the form of enhancing anexisting immune response or eliciting an initial immune response. Forexample, enhancing an immune response by modulation of ILT3 may beuseful in cases of viral infection. As the anti-ILT3 antibodies orantigen binding fragments disclosed herein may act to enhance immuneresponses, they would be therapeutically useful in situations where morerapid or thorough clearance of pathogenic agents, e.g., bacteria andviruses would be beneficial.

As used herein, the term “viral infection” includes infections withorganisms including, but not limited to, HIV (e.g., HIV-1 and HIV-2),human herpes viruses, cytomegalovirus (esp. Human), Rotavirus,Epstein-Barr virus, Varicella Zoster Virus, hepatitis viruses, such ashepatitis B virus, hepatitis A virus, hepatitis C virus and hepatitis Evirus, paramyxoviruses: Respiratory Syncytial virus, parainfluenzavirus, measles virus, mumps virus, human papilloma viruses (for exampleHPV6, 11, 16, 18 and the like), flaviviruses (e.g. Yellow Fever Virus,Dengue Virus, Tick-borne encephalitis virus, Japanese EncephalitisVirus) or influenza virus.

As used herein, the term “bacterial infections” include infections witha variety of bacterial organisms, including gram-positive andgram-negative bacteria. Examples include, but are not limited to,Neisseria spp, including N. gonorrhea and N. meningitidis, Streptococcusspp, including S. pneumoniae, S. pyogenes, S. agalactiae, S. mutans;Haemophilus spp, including H. influenzae type B, non typeable H.influenzae, H. ducreyi; Moraxella spp, including M. catarrhalis, alsoknown as Branhamella catarrhalis; Bordetella spp, including B.pertussis, B. parapertussis and B. bronchiseptica; Mycobacterium spp.,including M. tuberculosis, M. bovis, M. leprae, M. avium, M.paratuberculosis, M. smegmatis; Legionella spp, including L.pneumophila; Escherichia spp, including enterotoxic E. coli,enterohemorragic E. coli, enteropathogenic E. coli; Vibrio spp,including V. cholera, Shigella spp, including S. sonnei, S. dysenteriae,S. flexnerii; Yersinia spp, including Y. enterocolitica, Y. pestis, Y.pseudotuberculosis, Campylobacter spp, including C. jejuni and C. coli;Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S.enteritidis; Listeria spp., including L. monocytogenes; Helicobacterspp, including H. pylori; Pseudomonas spp, including P. aeruginosa,Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcusspp., including E. faecalis, E. faecium; Clostridium spp., including C.tetani, C. botulinum, C. difficile; Bacillus spp., including B.anthracis; Corynebacterium spp., including C. diphtheriae; Borreliaspp., including B. burgdorferi, B. garinii, B. afzelii, B. andersonii,B. hermsii; Ehrlichia spp., including E. equi and the agent of the HumanGranulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii;Chlamydia spp., including C. trachomatis, C. neumoniae, C. psittaci;Leptsira spp., including L. interrogans; Treponema spp., including T.pallidum, T. denticola, T. hyodysenteriae. Preferred bacteria include,but are not limited to, Listeria, mycobacteria, mycobacteria (e.g.,tuberculosis), Anthrax, Salmonella and Listeria monocytogenes.

In another embodiment, T cells can be removed from a patient, andcontacted in vitro with an anti-ILT3 antibody or antigen bindingfragment disclosed herein, optionally with an activating signal (e.g.,antigen plus APCs or a polyclonal antibody) and reintroduced into thepatient.

The anti-ILT3 antibodies or antigen binding fragments disclosed hereinmay also be used prophylactically in vaccines against various pathogens.Immunity against a pathogen, e.g., a virus, could be induced byvaccinating with a viral protein along with an anti-ILT3 antibody orantigen binding fragment disclosed herein. Alternately, an expressionvector that encodes genes for both a pathogenic antigen and anti-ILT3antibody or antigen binding fragment disclosed herein, e.g., a vacciniavirus expression vector engineered to express a nucleic acid encoding aviral protein and a nucleic acid encoding an anti-ILT3 antibody orantigen binding fragment disclosed herein, may be used for vaccination.Pathogens for which vaccines may be useful include, for example,hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1,HIV-2, tuberculosis, malaria and schistosomiasis.

The present invention further encompasses an anti-ILT3 antibody orantigen binding fragment disclosed herein conjugated to a diagnostic ortherapeutic agent. The anti-ILT3 antibody or antigen binding fragmentdisclosed herein can be used diagnostically to, for example, monitor thedevelopment or progression of a tumor as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.Detection may be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. The detectable substance may be coupled orconjugated either directly to the binding molecule or indirectly,through an intermediate (such as, for example, a linker known in theart) using techniques known in the art. U.S. Pat. No. 4,741,900discloses metal ions that may be conjugated to binding molecules.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive materials are ¹²⁵I, ¹³¹I, and ⁹⁹Tc.

Further, an anti-ILT3 antibody or antigen binding fragment disclosedherein may be conjugated to a therapeutic moiety such as a cytotoxin,e.g., a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi.A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, camustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The present invention is further directed to therapies that involveadministering an anti-ILT3 antibody or antigen binding fragmentdisclosed herein to an animal, preferably a mammal, and most preferablya human, patient for treating, detecting, and/or preventing one or moreof the diseases, disorders, or conditions disclosed herein. Therapeuticcompounds of the invention include, but are not limited to, anti-ILT3antibody or antigen binding fragment disclosed herein. The anti-ILT3antibody or antigen binding fragment disclosed herein may be used totreat, diagnose, inhibit or prevent diseases, disorders or conditionsassociated with aberrant activity of ILT3, including, but not limitedto, any one or more of the diseases, disorders, or conditions describedherein.

The anti-ILT3 antibody or antigen binding fragment disclosed herein maybe advantageously utilized in combination with other monoclonal orchimeric binding molecules, or with lymphokines or hematopoietic growthfactors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serveto increase the number or activity of effector cells which interact withthe binding molecules.

The anti-ILT3 antibody or antigen binding fragment disclosed herein maybe administered alone or in combination with other types of treatments,e.g., immunostimulatory treatments or treatments designed to control theproliferation of a target of activated immune cells (e.g., cancer cellsor pathogens). Exemplary therapies include e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents,antibiotics, and immunoglobulin.

An anti-ILT3 antibody or antigen binding fragment disclosed herein maybe administered to a human subject for therapeutic purposes. Moreover,an anti-ILT3 antibody or antigen binding fragment disclosed herein maybe administered to a non-human mammal expressing ILT3 with which thebinding molecule cross-reacts (e.g., a primate) for veterinary purposesor as an animal model of human disease.

Combinations

The anti-ILT3 antibodies or antigen binding fragments herein may be usedin unconjugated forms or conjugated to a second agent, e.g., a cytotoxicdrug, radioisotope, or a protein, e.g., a protein toxin or a viralprotein. This method includes: administering the anti-ILT3 antibodies orantigen binding fragments herein, alone or conjugated to a cytotoxicdrug, to a subject requiring such treatment. The anti-ILT3 antibodies orantigen binding fragments herein may be used to deliver a variety oftherapeutic agents, e.g., a cytotoxic moiety, e.g., a therapeutic drug,a radioisotope, molecules of plant, fungal, or bacterial origin, orbiological proteins (e.g., protein toxins) or particles (e.g., arecombinant viral particles, e.g.; via a viral coat protein), ormixtures thereof.

Additional Combination Therapies

The anti-ILT3 antibodies or antigen binding fragments herein may be usedin combination with other therapies. For example, the combinationtherapy may include a composition comprising an anti-ILT3 antibody orantigen binding fragment co-formulated with, and/or co-administeredwith, one or more additional therapeutic agents, e.g., one or moreanti-cancer agents, cytotoxic or cytostatic agents, hormone treatment,vaccines, and/or other immunotherapies. In other embodiments, theanti-ILT3 antibody or antigen binding fragment is administered incombination with other therapeutic treatment modalities, includingsurgery, radiation, cryosurgery, and/or thermotherapy. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

By “in combination with,” it is not intended to imply that the therapyor the therapeutic agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope described herein. The anti-ILT3 antibody or antigenbinding fragment may be administered concurrently with, prior to, orsubsequent to, one or more other additional therapies or therapeuticagents. The anti-ILT3 antibody or antigen binding fragment and the otheragent or therapeutic protocol may be administered in any order. Ingeneral, each agent will be administered at a dose and/or on a timeschedule determined for that agent. In will further be appreciated thatthe additional therapeutic agent utilized in this combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that additionaltherapeutic agents utilized in combination be utilized at levels that donot exceed the levels at which they are utilized individually. In someembodiments, the levels utilized in combination will be lower than thoseutilized individually.

In certain embodiments, an anti-ILT3 antibody or antigen bindingfragment described herein is administered in combination with one ormore check point inhibitors or antagonists of programmed death receptor1 (PD-1) or its ligand PD-L1 and PD-L2. The inhibitor or antagonist maybe an antibody, an antigen binding fragment, an immunoadhesin, a fusionprotein, or oligopeptide. In some embodiments, the anti-PD-1 antibody ischosen from nivolumab (OPDIVO®, Bristol Myers Squibb, New York, N.Y.),pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp, Kenilworth, N.J.USA), cetiplimab (Regeneron, Tarrytown, N.Y.) or pidilizumab (CT-011).In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence)). In some embodiments, the PD-1 inhibitor isAMP-224. In some embodiments, the PD-L1 inhibitor is anti-PD-L1 antibodysuch durvalumab (IMFINZI®, Astrazeneca, Wilmingon, Del.), atezolizumab(TECENTRIQ®, Roche, Zurich, CH), or avelumab (BAVENCIO®, EMD Serono,Billerica, Mass.). In some embodiments, the anti-PD-L1 bindingantagonist is chosen from YW243.55.S70, MPDL3280A, MEDI-4736,MSB-0010718C, or MDX-1105.

MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody describedin WO2007/005874. Antibody YW243.55.S70 is an anti-PD-L1 described in WO2010/077634 (heavy and light chain variable region sequences shown inSEQ ID NOs. 20 and 21, respectively).

Nivolumab, also known as OPDIVO®, MDX-1106-04, ONO-4538, or BMS-936558,is a fully human IgG4 anti-PD-1 antibody described in WO2006/121168 andU.S. Pat. No. 8,008,449.

Pembrolizumab, also known as KEYTRUDA®, lambrolizumab, MK-3475 orSCH-900475, is a humanized anti-PD-1 antibody described in U.S. Pat. No.8,354,509 and WO2009/114335 and disclosed, e.g., in Hamid, et al., NewEngland J. Med. 369 (2): 134-144 (2013). The heavy and light chains forprembrolizumab are shown by the amino acid sequences set forth in SEQ IDNos: 225 and 226, respectively.

Pidilizumab, also known as CT-011 (Cure Tech) is a humanized IgG1monoclonal antibody that binds to PD-1. Pidilizumab and other humanizedanti-PD-1 monoclonal antibodies are disclosed in WO2009/101611. Otheranti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g.,anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,609,089; U.S.Publication No. 2010028330; and U.S. Publication No. 20120114649.

AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 andWO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks theinteraction between PD-1 and B7-H1.

MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonalantibody that binds to PD-L1. MDPL3280A and other human monoclonalantibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.S.Publication No. 20120039906.

Other anti-PD-L1 binding agents include YW243.55.S70 (heavy and lightchain variable regions are shown in SEQ ID NOs 20 and 21 inWO2010/077634) and MDX-1105 (also referred to as BMS-936559). It andother anti-PD-L1 binding agents are disclosed in WO2007/005874).

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, the anti-ILT3 antibodies or antigenbinding fragments thereof, as discussed herein in association with oneor more additional components including, but not limited to, a furthertherapeutic agent, as discussed herein. The antibody or fragment and/orthe therapeutic agent can be formulated as a pure composition or incombination with a pharmaceutically acceptable carrier, in apharmaceutical composition.

In one embodiment, the kit includes the anti-ILT3 antibodies or antigenbinding fragments thereof or a pharmaceutical composition thereof in onecontainer (e.g., in a sterile glass or plastic vial) and a furthertherapeutic agent in another container (e.g., in a sterile glass orplastic vial).

In another embodiment, the kit comprises a combination of the anti-ILT3antibodies or antigen binding fragments thereof or pharmaceuticalcomposition thereof in combination with one or more therapeutic agentsformulated together, optionally, in a pharmaceutical composition, in asingle, common container.

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can include a device for performingsuch administration. For example, the kit can include one or morehypodermic needles or other injection devices as discussed above. Thus,the present invention includes a kit comprising an injection device andt the anti-ILT3 antibodies or antigen binding fragments thereof, e.g.,wherein the injection device includes the antibody or fragment orwherein the antibody or fragment is in a separate vessel.

The kit can include a package insert including information concerningthe pharmaceutical compositions and dosage forms in the kit. Generally,such information aids patients and physicians in using the enclosedpharmaceutical compositions and dosage forms effectively and safely. Forexample, the following information regarding a combination of theinvention may be supplied in the insert: pharmacokinetics,pharmacodynamics, clinical studies, efficacy parameters, indications andusage, contraindications, warnings, precautions, adverse reactions,overdosage, proper dosage and administration, how supplied, properstorage conditions, references, manufacturer/distributor information andpatent information.

Methods of Making Antibodies and Antigen Binding Fragments Thereof

The anti-ILT3 antibodies or antigen binding fragments thereof disclosedherein may also be produced recombinantly. In this embodiment, nucleicacid molecules encoding the antibody molecules may be inserted into avector (plasmid or viral) and transfected or transformed into a hostcell where it may be expressed and secreted from the host cell. Thereare several methods by which to produce recombinant antibodies which areknown in the art.

In particular aspects, the present invention provides nucleic acidmolecules encoding an HC and an LC wherein the HC comprises at least theHC-CDR3 of an anti-ILT3 antibody disclosed herein or embodiment thereofwherein the HC-CDR3 has one, two, or three amino acid substitutions,additions, deletions, or combinations thereof. In further embodiments,the HC and/or LC variable region framework comprises 0, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof.

In particular aspects, the present invention provides nucleic acidmolecules encoding an HC and an LC wherein the HC comprises the HC-CDR1,2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereofwherein one or more of HC-CDR1, 2, and 3 has one, two, or three aminoacid substitutions, additions, deletions, or combinations thereof andwherein the LC comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibodydisclosed herein or embodiment thereof wherein one or more of HC-CDR1,2, and 3 has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof. In further embodiments, the HCand/or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acid substitutions, additions, deletions, or combinationsthereof.

In particular aspects, the present invention provides a first expressionvector comprising a nucleic acid molecule encoding an HC comprising atleast the HC CDRs of an anti-ILT3 antibody disclosed herein orembodiment thereof wherein one or more of the three HC CDRs has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof and/or wherein the HC variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof and a second expressionvector comprising a nucleic acid molecule encoding an LC comprising atleast the LC CDRs of an anti-ILT3 antibody disclosed herein orembodiment thereof wherein one or more of the three LC CDRs has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof and/or wherein the LC variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acidmolecules encoding a V_(H) and a V_(L) wherein the V_(H) comprises atleast the HC-CDR3 of an anti-ILT3 antibody disclosed herein orembodiment thereof wherein the HC-CDR3 has one, two, or three amino acidsubstitutions, additions, deletions, or combinations thereof. In furtherembodiments, the V_(H) and/or V_(L) variable region framework comprises0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acidmolecules encoding a V_(H) and a V_(L) wherein the HC comprises theHC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein orembodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof and wherein the V_(L) comprises the LC-CDR1, 2, and3 of an anti-ILT3 antibody disclosed herein or embodiment thereofwherein one or more of HC-CDR1, 2, and 3 has one, two, or three aminoacid substitutions, additions, deletions, or combinations thereof. Infurther embodiments, the V_(H) and/or V_(L) variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acidmolecules encoding a V_(H) comprising at least the HC CDRs of ananti-ILT3 disclosed herein or embodiment thereof wherein one or more ofthe three HC CDRs has one, two, or three amino acid substitutions,additions, deletions, or combinations thereof and/or wherein the V_(H)and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof and nucleic acid molecules encoding a V_(L)comprising at least the LC CDRs of an anti-ILT3 antibody disclosedherein or embodiment thereof wherein one or more of the three LC CDRshas one, two, or three amino acid substitutions, additions, deletions,or combinations thereof and/or wherein the V_(H) and/or V_(L) variableregion framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid substitutions, additions, deletions, or combinations thereof.

Mammalian cell lines available as hosts for expression of the antibodiesor fragments disclosed herein are well known in the art and include manyimmortalized cell lines available from the American Type CultureCollection (ATCC). These include, inter alia, Chinese hamster ovary(CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, 3T3 cells, human embryo kidney 293 (HEK-293)cells and a number of other cell lines. Cell lines of particularpreference are selected through determining which cell lines have highexpression levels. Other cell lines that may be used are insect celllines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells,filamentous fungus cells (e.g. Trichoderma reesei), and yeast cells(e.g., Saccharomyces cerevisiae or Pichia pastoris). In particularaspects, the host cell may be a prokaryote host cell such as E. coli.

When recombinant expression vectors comprising a nucleic acid moleculeencoding the heavy chain or antigen-binding portion or fragment, thelight chain and/or antigen-binding fragment are introduced into hostcells, the antibodies are produced by culturing the host cells underconditions and for a period of time sufficient to allow for expressionof the antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown. Theantibodies may be recovered from the culture medium and further purifiedor processed to produce the antibodies of the invention.

In particular aspects, the host cells are transfected with an expressionvector comprising nucleic acid molecules encoding an HC and an LCwherein the HC comprises at least the HC-CDR3 of an anti-ILT3 antibodyor embodiment thereof wherein the HC-CDR3 has one, two, or three aminoacid substitutions, additions, deletions, or combinations thereof. Infurther embodiments, the HC and/or LC variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with an expressionvector comprising nucleic acid molecules encoding an HC and an LCwherein the HC comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibodydisclosed herein or embodiment thereof wherein one or more of HC-CDR1,2, and 3 has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof and wherein the LC comprises theLC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein orembodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof. In further embodiments, the HC and/or LC variableregion framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with a firstexpression vector comprising a nucleic acid molecule encoding an HCcomprising at least the HC CDRs of an anti-ILT3 antibody disclosedherein or embodiment thereof wherein one or more of the three HC CDRshas one, two, or three amino acid substitutions, additions, deletions,or combinations thereof and/or wherein the HC variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof and a second expressionvector comprising a nucleic acid molecule encoding an LC comprising atleast the LC CDRs of an antibody disclosed herein or embodiment thereofwherein one or more of the three LC CDRs has one, two, or three aminoacid s substitutions, additions, deletions, or combinations thereofand/or wherein the LC variable region framework comprises 0, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof.

In particular aspects, the host cells are transfected with an expressionvector comprising nucleic acid molecules encoding a V_(H) and a V_(L)wherein the V_(H) comprises at least the HC-CDR3 of an anti-ILT3antibody disclosed herein or embodiment thereof wherein the HC-CDR3 hasone, two, or three amino acid substitutions, additions, deletions, orcombinations thereof. In further embodiments, the V_(H) and/or V_(L)variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with an expressionvector comprising nucleic acid molecules encoding a V_(H) and a V_(L)wherein the V_(H) comprises the HC-CDR1, 2, and 3 of an anti-ILT3antibody disclosed herein or embodiment thereof wherein one or more ofHC-CDR1, 2, and 3 has one, two, or three amino acid substitutions,additions, deletions, or combinations thereof and wherein the V_(L)comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosedherein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3has one, two, or three amino acid substitutions, additions, deletions,or combinations thereof. In further embodiments, the V_(H) and/or V_(L)variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with a firstexpression vector comprising a nucleic acid molecule encoding a V_(H)comprising at least the HC CDRs of an anti-ILT3 antibody disclosedherein or embodiment thereof wherein one or more of the three HC CDRshas one, two, or three amino acid substitutions, additions, deletions,or combinations thereof and/or wherein the V_(H) variable regionframework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof and asecond expression vector comprising a nucleic acid molecule encoding aV_(L) comprising at least the LC CDRs of an anti-ILT3 antibody disclosedherein or embodiment thereof wherein one or more of the three LC CDRshas one, two, or three amino acid s substitutions, additions, deletions,or combinations thereof and/or wherein the V_(L) variable regionframework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof.

In particular embodiments, the HC and LC or V_(H) and V_(L) areexpressed as a fusion protein in which the N-terminus of the HC and theLC are fused to a leader sequence to facilitate the transport of theantibody through the secretory pathway. Examples of leader sequencesthat may be used include MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 12) orMEWSWVFLFFLSVTTGVHS (SEQ ID NO: 11).

The present invention further provides a plasmid or viral vectorcomprising a nucleic acid molecule encoding an anti-ILT3 antibodydisclosed herein or antigen binding fragment thereof. The presentinvention further provides a plasmid or viral vector comprising anucleic acid molecule encoding the HC of an anti-ILT3 antibody disclosedherein or antigen binding fragment thereof or embodiment of the antibodyor antigen binding fragment thereof wherein one or more of the threeCDRs has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof and/or wherein the HC variable regionframework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof and anucleic acid molecule encoding the LC of an anti-ILT3 antibody disclosedherein or antigen binding fragment thereof or embodiment of the antibodyor antigen binding fragment thereof wherein one or more of the threeCDRs has one, two, or three amino acid substitutions, additions,deletions, or combinations thereof and/or wherein the LC variable regionframework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof.

The present invention further provides a plasmid or viral vectorcomprising a nucleic acid molecule encoding the HC of an anti-ILT3antibody disclosed herein or antigen binding fragment thereof and aplasmid or viral vector comprising a nucleic acid molecule encoding theLC of an anti-ILT3 antibody disclosed herein or antigen binding fragmentthereof.

The present invention further provides a host cell comprising a plasmidor viral vector comprising a nucleic acid molecule encoding the HC of ananti-ILT3 antibody disclosed herein or antigen binding fragment thereofor embodiment of an anti-ILT3 antibody disclosed herein or antigenbinding fragment thereof wherein one or more of the three CDRs has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof and/or wherein the HC variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof and a plasmid or viralvector comprising a nucleic acid molecule encoding the LC of ananti-ILT3 antibody disclosed herein or antigen binding fragment thereofor embodiment of an anti-ILT3 antibody disclosed herein or antigenbinding fragment thereof wherein one or more of the three CDRs has one,two, or three amino acid substitutions, additions, deletions, orcombinations thereof and/or wherein the LC variable region frameworkcomprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,additions, deletions, or combinations thereof. In particularembodiments, the host cell is a CHO or HEK-293 host cell.

The present invention further provides a plasmid or viral vectorcomprising a nucleic acid molecule encoding an anti-ILT3 antibodydisclosed herein or antigen binding fragment thereof. The presentinvention further provides a plasmid or viral vector comprising anucleic acid molecule encoding the V_(H) of an anti-ILT3 antibodydisclosed herein or antigen binding fragment thereof or embodiment ofthe antibody or antigen binding fragment thereof wherein one or more ofthe three CDRs has one, two, or three amino acid substitutions,additions, deletions, or combinations thereof and/or wherein the V_(H)framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof and anucleic acid molecule encoding the V_(L) of an anti-ILT3 antibodydisclosed herein or antigen binding fragment thereof or embodiment ofthe antibody or antigen binding fragment thereof wherein one or more ofthe three CDRs has one, two, or three amino acid substitutions,additions, deletions, or combinations thereof and/or wherein the LCframework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions, additions, deletions, or combinations thereof.

The present invention further provides a plasmid or viral vectorcomprising a nucleic acid molecule encoding the V_(H) of an anti-ILT3antibody disclosed herein or antigen binding fragment thereof and aplasmid or viral vector comprising a nucleic acid molecule encoding theV_(L) of an anti-ILT3 antibody disclosed herein or antigen bindingfragment thereof.

The present invention further provides a host cell comprising a plasmidor viral vector comprising a nucleic acid molecule encoding the V_(H) ofan anti-ILT3 antibody disclosed herein or antigen binding fragmentthereof or embodiment of an anti-ILT3 antibody disclosed herein orantigen binding fragment thereof wherein one or more of the three CDRshas one, two, or three amino acid substitutions, additions, deletions,or combinations thereof and/or wherein the V_(H) framework comprises 0,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,deletions, or combinations thereof and a plasmid or viral vectorcomprising a nucleic acid molecule encoding the V_(L) of an anti-ILT3antibody disclosed herein or antigen binding fragment thereof orembodiment of an anti-ILT3 antibody disclosed herein or antigen bindingfragment thereof wherein one or more of the three CDRs has one, two, orthree amino acid substitutions, additions, deletions, or combinationsthereof and/or wherein the V_(L) framework comprises 0, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, orcombinations thereof. In particular embodiments, the host cell is a CHOor HEK-293 host cell.

The anti-ILT3 antibodies or antigen binding fragments thereof can berecovered from the culture medium using standard protein purificationmethods. Further, expression of antibodies of the invention (or othermoieties therefrom) from production cell lines can be enhanced using anumber of known techniques. For example, the glutamine synthetase geneexpression system (the GS system) is a common approach for enhancingexpression under certain conditions.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal (See for example, Croset et al., J. Biotechnol. 161: 336-348(2012)). Therefore, the particular glycosylation pattern of an antibodywill depend on the particular cell line or transgenic animal used toproduce the antibody. However, all antibodies encoded by the nucleicacid molecules provided herein, or comprising the amino acid sequencesprovided herein, comprise the instant invention, independent of theglycosylation pattern the antibodies may have.

The following examples are intended to promote a further understandingof the present invention.

GENERAL METHODS

Standard methods in molecular biology are described Sambrook, Fritschand Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu(1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).Standard methods also appear in Ausbel, et al. (2001) Current Protocolsin Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York,N.Y., which describes cloning in bacterial cells and DNA mutagenesis(Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugatesand protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesare described (Coligan, et al. (2001) Current Protocols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al.(1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote andWinter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol.17:397-399).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes,liposomes, polyethylene glycol (PEG). Antibodies are useful fortherapeutic, diagnostic, kit or other purposes, and include antibodiescoupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g.,colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol.146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J.Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cellsorting (FACS), are available (see, e.g., Owens, et al. (1994) FlowCytometry Principles for Clinical Laboratory Practice, John Wiley andSons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss,Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, John Wiley andSons, Hoboken, N.J.). Fluorescent reagents suitable for modifyingnucleic acids, including nucleic acid primers and probes, polypeptides,and antibodies, for use, e.g., as diagnostic reagents, are available(Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene,Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see,e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology andPathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) ColorAtlas of Histology, Lippincott, Williams, and Wilkins, Phila, P A;Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, NewYork, N.Y.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GenBank, VECTOR NTI® Suite (Informax, Inc, Bethesda, Md.); GCG WisconsinPackage (Accelrys, Inc., San Diego, Calif.); DECYPHER® (TimeLogic Corp.,Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742;Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren,et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.14:4683-4690).

Purity determinations: Size-exclusion ultra-high performance liquidchromatography (SE-UPLC) or (SEC) was carried out on an ACQUITY® UPLC®H-Class system. Column used was an ACQUITY® UPLC® Protein BEH SEC column(Part No. 186005225, 1.7 μm, 200 Å, 4.6 mm×150 mm) from Waters (Milford,Mass.). Column temperature used was 25 C and 10 μl sample at 1 mg/mL wasinjected using a system flow rate of 0.5 ml/min. Mobile phase used was100 mM sodium phosphate, 200 mM sodium chloride and 0.02% sodium azide,pH 7.0. Data was quantified at both 214 and 280 nm and analyzed usingEmpower 3 software. A BEH200 SEC Protein Standard Mix (Part No.186006518) from Waters (Milford, Mass.) was utilized and injected at 10ug and USP Resolution, Theoretical plates, and Tailing was measured.

NANO-DSF™ (tradename for modified differential scanning fluorimetrymethod to determine protein stability employing intrinsic tryptophan ortyrosin fluorescence): the temperature mid-point of a thermal unfoldingcurve, Tm, and mid-point of a thermal aggregation curve, Tagg, weredetermined by NANO-DSF™ using a PROMETHEUS™ NT.48 Differential ScanningFluorimeter (Nanotemper Technologies) controlled by PR THERMCONTROL™v2.0.4 software. Excitation power was 40% and temperature was increasedfrom 20° C. to 95° C. at a rate of 1 C/minute. Tm and Tagg wereautomatically measured. Samples were prepared by diluting to 1 mg/mL in20 mM sodium acetate pH 5.5 buffer and drawn by capillary action into aPROMETHEUS™ glass capillary (PR-L002).

Capillary Isoelectric Focusing (cIEF): cIEF was conducted on a iCE3™system from Protein Simple (San Jose, Calif.) using iCE CFR™ software4.1.1 for instrument control and data analysis. cIEF Cartridge used wasFc-coated (Protein Simple, 101701) and prepared according tomanufacturer's instruction. A 200 μL sample consisting of 40 μg ofanalyte and 1% v/v 3-10 PHARMALYTE®, 0.5% v/v 8-10.5 PHARMALYTE®, 0.5%v/v 5-8 PHARMALYTE® (GE Healthcare), 37.5% v/v 8.0 M Urea(Sigma-Aldrich), 35% v/v 1% methyl cellulose and 1 μL each of 5.85 and9.22 pI markers (Protein Simple), was prepared. Samples were injectedfor 60 seconds. Isoelectric focusing parameters were 1500 V for 1 minuteand 3000 V for 8 minutes. pI was automatically measured using theinternal pI markers serving as a two-point calibration standard.Calibrated data was further analyzed and quantified by conversion toEmpower format and analyzed using Empower 3.

Example 1

Hybridoma clone 52B8 was identified via standard mouse and ratimmunization and hybridoma selections. In general, Balb/C mice or ratswere immunized with human ILT3-HIS recombinant protein in a standardfour week footpad immunization to generate a hyperimmune response.Electrofusion of bulk lymphocytes from draining lymph nodes with the P3myeloma fusion partner produced immortalized hybridomas. Hybridomasupernatant fluid was screened in a primary cell-based ELISA bindingassay on human CHO-human ILT3 cells. A secondary screen on CHO parental,CHO-ILT3 SNP, CHO-rhesus ILT3, CHO-ILT5, CHO-ILT8, and CHO-ILT11 cellswas performed in a cell-based ELISA format (See Example 2). Subcloningby limited dilution was performed on the ILT3 specific and rhesuspositive hybridoma cells. Subclones were expanded to generate purifiedprotein to enable additional tests of Biacore analyses and functionalscreening. Table 5 shows 10 hybridoma clones that produced antibodiesthat binned together and had high affinity for human ILT3 as shown byCELISA and Biacore preformed as disclosed in Examples 2 and 4,respectively.

TABLE 5 cELISA- human cELISA- ILT3 rhesus ILT3 Biacore Kd Parental EC50EC50 Biacore Kd (M)- Clone species (ng/mL) (ng/mL) (M)-ILT3_H ILT3_MMLB181.52A8.1A1 Mouse 18.4 25  8.55 × 10⁻¹⁰  1.3 × 10⁻⁸ LB181.52B8.1B1Mouse 15.5 23.2  6.58 × 10⁻¹⁰ 2.44 × 10⁻⁸ LB182.11D1.1A1 Mouse 50.5 NoBinding  1.41 × 10⁻⁰⁸ No binding LB182.1G12.1B1 Mouse 39.2 No Binding 1.69 × 10⁻⁰⁸ No binding LB184.16B1.1D2 Rat 64.9 67.9  9.57 × 10⁻¹¹ 2.59× 10⁻¹⁰ LB184.20E4.1E1.1D1 Rat 2 18  6.99 × 10⁻⁹  1.8 × 10⁻⁸LB184.24A4.1A1 Rat 21.4 23.1  2.05 × 10⁻¹¹ 1.26 × 10⁻¹⁰LB184.37C8.1A3.1B1 Rat 7.7 9.5 1.18. × 10⁻¹¹  1.5 × 10⁻¹⁰ LB184.40A6.1C1Rat 17.9 25.9  1.79 × 10⁻⁰⁹ 9.46 × 10⁻¹⁰ LB190.17H12.1A1 Rat 139.2 NoBinding  5.92 × 10⁻¹⁰ No binding H = human MM = rhesus monkey (Macacamulatta)Table 6 shows the amino acid sequences for the heavy chain and lightchain variable domains for the mAbs obtained from the above clones.

TABLE 6 SEQ ID NO: Heavy Chain Light Chain mAb Variable Variable No.Description domain Domain p52B8 Mouse anti-ILT3 mAb 52B8 IgG2a/Kappa  15 16 p40A6 Rat anti-ILT3 mAb 40A6 IgG2a/Kappa  45  46 p16B1 Rat anti-ILT3mAb 16B1 IgG2a/Kappa  53  54 p49C6 Mouse anti-ILT3 mAb 49C6 IgG2a/KappaNot sequenced Not sequenced p11D1 Mouse anti-ILT3 mAb 11D1 IgG2b/Kappa 61  62 p17H12 Rat anti-ILT3 mAb 17H12 IgG1/Kappa  69  70 p37C8 Ratanti-ILT3 mAb 37C8 IgG2a/Kappa  77  78 p1G12 Mouse anti-ILT3 mAb 1G12IgG2a/Kappa  85  86 p20E4 Rat anti-ILT3 mAb 20E4 IgG2a/Kappa  93  94p24A4 Rat ant-ILT3 mAb 24A4 IgG2a/Kappa 101 102

To ultimately guide the selection of a lead antibody, antibodies werefurther analyzed and re-evaluated in a set of bio-functional,biophysical, and physicochemical assays. Finally, antibodies were testedin an in vivo, proof of biology tumor regression study using humanSKMEL5 melanoma-challenged humanized mice.

Example 2 Selectivity of Various Anti-ILT3 Antibodies

Cell-based ELISA (cELISA) was used to show the selectivity of thevarious parental anti-ILT3 antibodies shown in Table 5 and humanizedanti-ILT3 monoclonal antibody 9B11 disclosed in U.S. Pat. No. 7,777,008as having the amino acid sequences of SEQ ID NO: 33 (light chain) andSEQ ID NO: 34 (heavy chain).

Mouse anti-human ILT3 antibodies were tested for binding to human ILT3,and cross-reactivity to Rhesus monkey ILT3, human ILT5, human ILT7,human ILT8, and human ILT11 expressing CHO-K1 cells using a cell-basedELISA format. CHO-K1 cells were plated in 96-well tissue-culture platesin 50 μL of DMEM/F12, 10% BCS and gentamycin (CHO-K1 media). Cells wereplated at either 2×10⁴ cells/well two days prior to the assay or 4×10⁴cells/well one day prior to the assay. Media was removed from the wellsprior to adding the test samples. Purified antibody was serially-dilutedin CHO-K1 media and added to the CHO-K1 plates. The samples wereincubated at room temperature for 30-60 minutes and plates were washedthree times with PBS/05% Tween-20 using the cell wash program on theBiotek EL405x Select CW plate washer. Binding was detected using anHRP-conjugated goat anti-mouse IgG (Southern Biotech cat #1031-05)secondary antibody added at a 1:2000 dilution in CHO-K1 media andincubated at room temperature for 30-60 minutes. Assay plates werewashed as above and developed with TMB and stopped with TMB stopsolution (KPL cat #50-85-06). The absorbance at 450 nm-620 nm wasdetermined. Mouse IgG1 (MIgG1) served as a control

The results are shown in FIGS. 1A, 1B, 1C, 1D, and 1E. The figures showthat representative antibodies from clones p40B5, p49C6, and p52B8 werespecific for ILT3 and did not cross-react with or bind ILT5, ILT7, ILT8,and ILT11. Antibodies from clones p49C6 and p52B8 as were the antibodiesfrom the other clones were capable of binding Rhesus monkey ILT3. Thep52B8 clone was chosen for in vivo characterization based on (1) itshigh affinity to human ILT3, (2) lack of binding to other ILT familymembers, and (3) cross-reactivity to rhesus ILT3.

Example 3

Parental mouse 52B8 heavy chain (VH) and light chain (VL) variabledomain sequences were compared to human germline sequences. Humanframework sequences closely homologous to the framework of the mouseantibody were chosen.

The mouse V_(H) domain of mouse anti-human ILT3 mAb 52B8 clone scoredhighly against human heavy chain germline 3-07 in subgroup III and JH4for the J region. Based on structural considerations, two frameworksubstitutions (R87K and A97G) were incorporated to maintain bindingequivalent to the parental antibody. The mouse V_(L) domain of theantibody clone scored highly against human light chain germline 1-O2 inkappa subgroup I. Mouse 52B8 CDRs were engineered onto the variablelight chain sequence of 1-O2 and JK2 for the J region. Based onstructural considerations, three framework substitutions (M4L, S64A, andG72R) were incorporated.

To generate humanized variants, the humanized V_(H) sequence was clonedinto a vector encoding human IgG4 S228P heavy chain constant domain andthe humanized V_(L) domain was cloned into a vector encoding for a kappalight chain constant domain. A total of two humanized V_(H) (VH1 andVH2) and 8 humanized V_(L) were designed. In silico sequence andstructural analysis of mouse 52B8 revealed six potential “hot spots” onthe molecule: two potential oxidation sites in VH-CDR2 (M64) and inVH-CDR3 (W101), one potential isomerization site in VH-CDR2 (D62), onepotential deamidation site in VL-CDR1 (N34), two potential isomerizationsites in VL-CDR1 (D30) and VL-CDR2 (D59). M64 was modified to V64 orL64, which maintained favorable physicochemical attributes andbinding/functionality.

FIG. 2A provides a table showing data characteristics on bindingaffinity, isoelectric point, purity of monomer species, and thermalstability measurements for humanized variants that were designed.Biacore was used to measure binding affinity, cIEF was used to measurepI, purity was determined by SE-UPLC, Tm and Tgg was determined byNANO-DSF™. FIG. 2B shows the relationship of SEC purity and meltingtemperature of various humanized light chain variants. Data is plottedas values obtained from each of the eight humanized light chain variantsdemonstrating that VL5 has both the highest purity and thermalstability. Based on the data in FIG. 2A and FIG. 2B, VL5 was selectedfor the light chain.

Initial studies were performed on the humanized VH1 M64V/VL5 produced intransient CHO cells. Forced deamidation conditions employing both 50° C.incubation and high pH stress performed on unformulated humanized 52B8VH1 M64V/VL5 revealed deamidation of LC N34 in VL-CDR1 (4.0 and 7.2%,respectively) and W101 oxidation in HC-CDR3 with 1× light stressexposure was 15.4%. Substitution of N34 to Q34 maintained bindingaffinity to human and rhesus ILT3 assessed by a Biacore SPR assay andfunctional activity assessed by a DC TNFα production assay; however,substitution of the W101 residue resulted in significant loss in bindingas determined by a Biacore SPR assay.

In summary, the humanized 52B8 was anti-ILT3 mAb (52B8 VH1 M64V/VL5 N34QIgG4 S228P/Kappa), contains one framework substitution in V_(L) (M4L)and one framework substitution in V_(H) (A97G).

Example 4 Binding Kinetics and Affinities for the Anti-Human ILT3Antibodies to Recombinant Human or Rhesus ILT3

The binding kinetics and affinities of anti-human ILT3 clones for humanor rhesus ILT3-His tagged recombinant protein were measured by surfaceplasmon resonance using a Biacore T200 system (GE Healthcare,Piscataway, N.J.). HBS-EP+ buffer (BR-1006-69) was used as the runningbuffer. Anti-human Fc antibody (Human Fc Capture Kit, BR100839, GEHealthcare) was immobilized via amine coupling chemistry in all fourflow cells on a Series S CM5 sensor chip (BR100530 or 29149603, GEHealthcare) following manufacturer instructions. Flow cell 1 was used asreference for background subtraction and was not used for capture.Anti-human ILT3 antibodies listed above (diluted to 1 μg/mL in HBS-EP+buffer) were injected over the anti-human Fc capture surfaces in flowcells 2, 3 and 4 at 10 μL/mL for 10 seconds which resulted in antibodycapture levels in the range of 60-70 RU Six-point, two-fold dilutionseries of human or rhesus ILT3-His protein ranging from 20 nM to 0.31 nMand two zeros (HBS-EP+) were injected at 50 μL/mL over the reference andcaptured antibody surfaces for 180 seconds of association followed by600 seconds of dissociation. Following each injection cycle, all fourflow cells were regenerated using 30 second injection of 3M MgCl₂solution at a flow rate of 10 μL/minute. Reference subtractedsensorgrams were fit to a 1:1 Langmuir Binding Model in the Biacore T200Evaluation Software (Version 2.0) to determine the association (ka) anddissociation (kd) rate constants and the equilibrium dissociationconstant KD (=kd/ka).

Table 7 summarizes the binding kinetics and affinities for theanti-human ILT3 antibodies to recombinant human or rhesus ILT3.

TABLE 7 cELISA cELISA Biacore Biacore (human (rhesus KD KD Purity ILT3-ILT3- (human (rhesus by SEC CHO) CHO) ILT3- ILT3- (% mAb EC50 EC50 His)His) main No. Description (μg/mL) (μg/mL) (nM) (nM) peak) pI 63 Chimericanti- 0.064 0.091 0.46 9.5 95.9 n.d. ILT3 52B8 mouse VH/human IgG4(S228P):mouse VL/human Kappa 64 Chimeric anti- 0.075 0.096 0.44 9.2 95.3n.d. ILT3 52B8 mouse VH M64V/human IgG4 (S228P):mouse VL/human Kappa 65Chimeric anti- 0.086 0.137 0.41 9.3 93.5 n.d. ILT3 52B8 mouse VHM64L/human IgG4 (S228P):mouse VL/human Kappa 1 Humanized anti- n.d. n.d.0.99 25 93.1 n.d. ILT3 mAb (52B8 VH1/VL1) IgG4 S228P/ Kappa 2 Humanizedanti- 0.7 0.109 1.1 20 96.2 n.d. ILT3 mAb (52B8 VH1/VL2) IgG4 S228P/Kappa 3 Humanized anti- n.d. n.d. 1.1 26 90 n.d. ILT3 mAb (52B8 VH1/VL3)IgG4 S228P/ Kappa 4 Humanized anti- n.d. n.d. 1.4 29 93.3 n.d. ILT3 mAb(52B8 VH1/VL4) IgG4 S228P/ Kappa 5 Humanized anti- n.d. n.d. 0.94 2593.1 n.d. ILT3 mAb (52B8 VH2/VL1) IgG4 S228P/ Kappa 6 Humanized anti-0.1 0.118 1.1 21 96.6 n.d. ILT3 mAb (52B8 VH2/VL2) IgG4 S228P/ Kappa 7Humanized anti- n.d. n.d. 0.96 26 89.6 6.33 ILT3 mAb (52B8 VH2/VL3) IgG4S228P/ Kappa 8 Humanized anti- n.d. n.d. 1.3 27 92.8 n.d. ILT3 mAb (52B8VH2/VL4) IgG4 S228P/ Kappa 9 Humanized anti- n.d. n.d. 0.94 26 92.1 n.d.ILT3 mAb (52B8 VH1 M64V/ VL1) IgG4 S228P/Kappa 10 Humanized anti- 0.0850.148 1.1 22 95.1 n.d. ILT3 mAb (52B8 VH1 M64V/ VL2) IgG4 S228P/Kappa 11Humanized anti- n.d. n.d. 1.1 27 89.6 n.d. ILT3 mAb (52B8 VH1 M64V/ VL3)IgG4 S228P/Kappa 12 Humanized anti- n.d. n.d. 1.5 29 92.4 n.d. ILT3 mAb(52B8 VH1 M64V/ VL4) IgG4 S228P/Kappa 13 Humanized anti- n.d. n.d. 0.9425 85.9 n.d. ILT3 mAb (52B8 VH2 M64V/ VL1) IgG4 S228P/Kappa 14 Humanizedanti- 0.077 0.126 1 22 92.8 n.d. ILT3 mAb (52B8 VH2 M64V/ VL2) IgG4S228P/Kappa 15 Humanized anti- n.d. n.d. 1 26 88.7 n.d. ILT3 mAb (52B8VH2 M64V/ VL3) IgG4 S228P/Kappa 16 Humanized anti- n.d. n.d. 1.4 29 93n.d. ILT3 mAb (52B8 VH2 M64V/ VL4) IgG4 S228P/Kappa 17 Humanized anti-n.d. n.d. 0.87 24 90.2 n.d. ILT3 mAb (52B8 VH1 M64L/ VL1) IgG4S228P/Kappa 18 Humanized anti- 0.079 0.137 1 22 92.2 n.d. ILT3 mAb (52B8VH1 M64L/ VL2) IgG4 S228P/Kappa 19 Humanized anti- n.d. n.d. 0.99 2687.4 n.d. ILT3 mAb (52B8 VH1 M64L/ VL3) IgG4 S228P/Kappa 20 Humanizedanti- n.d. n.d. 1.3 29 90.8 n.d. ILT3 mAb (52B8 VH1 M64L/ VL4) IgG4S228P/Kappa 21 Humanized anti- 0.079 0.112 0.88 27 91.2 n.d. ILT3 mAb(52B8 VH2 M64L/ VL1) IgG4 S228P/Kappa 22 Humanized anti- 0.057 0.0810.97 21 96.8 n.d. ILT3 mAb (52B8 VH2 M64L/ VL2) IgG4 S228P/Kappa 23Humanized anti- n.d. n.d. 0.96 24 88.5 n.d. ILT3 mAb (52B8 VH2 M64L/VL3) IgG4 S228P/Kappa 24 Humanized anti- n.d. n.d. 1.2 27 91.9 n.d. ILT3mAb (52B8 VH2 M64L/ VL4) IgG4 S228P/Kappa 25 Humanized anti- n.d. n.d.0.74 8.7 94.9 7.76 ILT3 mAb ((52B8 VH1 M64V/VL2) L234A L235A D265S)IgG1/ Kappa 26 Humanized anti- n.d. n.d. 0.61 4.9 96.05 8.62 ILT3 mAb((52B8 VH1 M64V/VL5) L234A L235A D265S) IgG1/ Kappa 27 Humanized anti-n.d. n.d. 0.92 10 90.17 8.84 ILT3 mAb ((52B8 VH1 M64V/VL6) L234A L235AD265S) IgG1/ Kappa 28 Humanized anti- n.d. n.d. 0.57 5.6 94.4 8.8 ILT3mAb ((52B8 VH1 M64V/VL7) L234A L235A D265S) IgG1/ Kappa 29 Humanizedanti- n.d. n.d. 0.56 5.7 94.14 8.85 ILT3 mAb ((52B8 VH1 M64V/VL8) L234AL235A D265S) IgG1/ Kappa 30 Humanized anti- n.d. n.d. 0.60 4.8 98.227.21 ILT3 mAb (52B8 VH1 M64V/ VL5) IgG4 S228P/Kappa 31 Humanized anti-n.d. n.d. 0.88 10 91.74 7.45 ILT3 mAb (52B8 VH1 M64V/ VL6) IgG4S228P/Kappa 32 Humanized anti- n.d. n.d. 0.53 5.6 97.79 7.45 ILT3 mAb(52B8 VH1 M64V/ VL7) IgG4 S228P/Kappa 33 Humanized anti- n.d. n.d. 0.545.6 97.29 7.45 ILT3 mAb (52B8 VH1 M64V/ VL8) IgG4 S228P/Kappa 34Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64VW101F/VL2) IgG4 S228P/ Kappa 35 Humanized anti- n.d. n.d. n.d. n.d. n.d.n.d. ILT3 mAb (52B8 VH1 M64V W101Y/VL2) IgG4 S228P/ Kappa 36 Humanizedanti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Q/VL2)IgG4 S228P/ Kappa 37 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3mAb ((52B8 VH1 M64V W101F/ VL2) L234A L235A D265S) IgG1/Kappa 38Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb ((52B8 VH1 M64VW101Y/ VL2) L234A L235A D265S) IgG1/Kappa 39 Humanized anti- n.d. n.d.n.d. n.d. n.d. n.d. ILT3 mAb ((52B8 VH1 M64V W101Q/ VL2) L234A L235AD265S) IgG1/Kappa 40 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3mAb (52B8 VH1 M64V/ VL2 S35A) IgG4 S228P/Kappa 41 Humanized anti- n.d.n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL2 S35N) IgG4S228P/Kappa 42 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb(52B8 VH1 M64V/ VL2 N34Q) IgG4 S228P/ Kappa 43 Humanized anti- n.d. n.d.n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL2 N34D) IgG4 S228P/ Kappa44 Humanized anti- n.d. n.d. 2.6 34 n.d. n.d. ILT3 mAb (52B8 VH1 M64V/VL5 S35A) IgG4 S228P/Kappa 45 Humanized anti- n.d. n.d. 4.7 NB n.d. n.d.ILT3 mAb (52B8 (No VH1 M64V/ Binding) VL5 S35N) IgG4 S228P/Kappa 46Humanized anti- 0.088 0.12 0.77 15 97.9 7.1 ILT3 mAb (52B8 VH1 M64V/ VL5N34Q) IgG4 S228P/ Kappa 47 Humanized anti- n.d. n.d. 3.8 115 n.d. n.d.ILT3 mAb (52B8 VH1 M64V/ VL5 N34D) IgG4 S228P/ Kappa 48 Humanized anti-n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101F/VL5) IgG4S228P/ Kappa 49 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb(52B8 VH1 M64V W101Y/VL5) IgG4 S228P/ Kappa 50 Humanized anti- n.d. n.d.n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Q/VL5) IgG4 S228P/ Kappa51 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1M64V Binding) Binding) W101F/VL5 S35A) IgG4 S228P/Kappa 52 Humanizedanti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding)Binding) W101F/VL5 S35N) IgG4 S228P/Kappa 53 Humanized anti- n.d. n.d.35 NB n.d. n.d. ILT3 mAb (52B8 (No VH1 M64V Binding) W101F/VL5 N34Q)IgG4 S228P/Kappa 54 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb(52B8 (No (No VH1 M64V Binding) Binding) W101F/VL5 N34D) IgG4S228P/Kappa 55 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8(No (No VH1 M64V Binding) Binding) W101Y/VL5 S35A) IgG4 S228P/Kappa 56Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1M64V Binding) Binding) W101Y/VL5 S35N) IgG4 S228P/Kappa 57 Humanizedanti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding)Binding) W101Y/VL5 N34Q) IgG4 S228P/Kappa 58 Humanized anti- n.d. n.d.NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding)W101Y/VL5 N34D) IgG4 S228P/Kappa 59 Humanized anti- n.d. n.d. NB NB n.d.n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Q/VL5 S35A)IgG4 S228P/Kappa 60 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb(52B8 (No (No VH1 M64V Binding) Binding) W101Q/VL5 S35N) IgG4S228P/Kappa 61 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8(No (No VH1 M64V Binding) Binding) W101Q/VL5 N34Q) IgG4 S228P/Kappa 62Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1M64V Binding) Binding) W101Q/VL5 N34D) IgG4 S228P/Kappa p52B8 Clone 52B815.5 23.2 0.658 24.4 98 n.d. Hybridoma extract p40A6 Clone 40A6 17.925.9 0.713 0.995 n.d. n.d. Hybridoma extract p16B1 Clone 16B1 n.d. n.d.0.096 0.259 98.1 n.d. Hybridoma extract p49C6 Clone 49C6 13.8 19.8 n.d.n.d. n.d. n.d. Hybridoma extract (not sequenced) p11D1 Clone 11D1 50.462028 n.d. n.d. n.d. n.d. Hybridoma extract p17H12 Clone 17H12 139.2 NBn.d. n.d. 95.7 n.d. Hybridoma extract p37C8 Clone 37C8 7.719 9.478 0.0120.145 98.4 n.d. Hybridoma extract p1G12 Clone 1G12 39.2 NB n.d. n.d.n.d. n.d. Hybridoma extract p20E4 Clone 20E4 1.992 18.04 6.99 18.2 98.5n.d. Hybridoma extract p24A4 Clone 24A4 21.4 21.3 0.021 0.126 n.d. n.d.Hybridoma extract

Example 5 Epitope Mapping of a Chimeric Anti-ILT3 52B8 Mouse VH/HumanIgG4 (S228P):Mouse VL/Human Kappa (“c58B8”; mAb 73) Binding to HumanILT3 by Hydrogen Deuterium Exchange (HDX) Mass Spectrometry

Contact areas of the antibody to human ILT3 extracellular domain weredetermined by use of hydrogen deuterium exchange mass spectrometry(HDX-MS) analysis. HDX-MS measures the incorporation of deuterium intothe amide backbone of the protein and changes in this incorporation areinfluenced by the hydrogen's solvent exposure. A comparison of thedeuterium exchange levels in antigen-alone samples and antibody-boundsamples was done to identify regions on the ILT3 extracellular domainthat may be in contact with the antibody. Human ILT3 extracellulardomain with a C-terminal His tag (human ILT3-His) has the amino acidsequence shown in SEQ ID NO: 1.

His-tagged human ILT3-His extracellular domain was pre-incubated withantibody c58B8 (mAb 73), a chimeric anti-ILT3 52B8 mouse VH M64V/humanIgG4 (S228P):mouse VL/human Kappa comprising a HC having the amino acidsequence of SEQ ID NO: 113 and a LC having the amino acid sequence shownin SEQ ID NO: 116, before incubation in a deuterium buffer. HumanILT3-His and the antibody were buffer exchanged to PBS pH 7.4 using 3 kMWCO spin columns. Human ILT3-His (80 pmol/μL) was mixed with an equalvolume of the antibody (40 pmol/μL) or, as the unbound control, PBS pH7.4. The antibody bound samples and the unbound control were incubatedat room temperature for one hour before beginning the labelingexperiment.

To deuterium label the samples, 2 μL of sample was mixed with 25 μL ofPBS in deuterium oxide pH 7.6. Labeling time points were 30, 300, 3000,6000 or 12000 seconds. After the set time, 25 μL of the labeling mixturewas added to 30 μL of cold quench buffer (8M Urea, 150 mM TCEP). Thequenched sample was incubated at 1.5° C. for 2 minutes. 53 μL was theninjected into the column cooling chamber where the sample was passedover the pepsin/protease XIII column and the resulting peptides loadedonto the trapping column. After three minutes, the analytical gradientand the mass spectrometer were started. A fully deuterated sample wasgenerated by incubating 2 μL of human ILT3-His with 108 μL of deuterateddenaturing buffer (4M Urea, 150 mM TCEP in 99.5% deuterium oxide). Thesample was incubated at 37° C. overnight. Then 55 μL was directlyinjected into the column chamber and the data acquired.

LC-MS/MS data was acquired of an unlabeled sample and searched beforedeuterium labeling to verify successful digestion of the proteins and togenerate a list of peptides. Data was database searched using ProteomeDiscoverer 1.4 and the SEQUEST HT search algorithm (ThermoFisherScientific). The protein database used was the human ILT3-His sequenceconcatenated to the yeast Saccharomycese cerevisiae database.

Following labeling, 55 μL sample aliquotes were applied to aNovaBioAssays Pepsin/Protease XIII column followed by chromatography onWaters CSH C18 Guard column and Waters CSH C18 1×50 mm Analytical columnin a loading buffer containing 2% Acetonitrile, 0.1% TFA. Deuteriumincorporation into the human ILT3-His extracellular domain was measuredby mass spectrometry. Quench: 8M Urea, 150 mM TCEP; Labeling buffer:PBS, pH 7.6; Blank buffer: PBS, pH 7.4. The mass spectrometer was aThermo Scientific ORBITRAP-ELITE™. For the measurement of deuteriumlabeled samples, the mass spectrometer was set to acquire one full scanMS data in the orbitrap at 120,000 resolving power, a target ion countof 1E6 and a maximum ion injection time of 500 millisecond. For theacquisition of MS/MS data for peptide identifications, the massspectrometer was set to acquire one full scan spectrum at 120,000resolving power followed by ten data-dependent MS/MS spectra in the iontrap.

The liquid chromatography system used was a Waters NANOACQUITY® for theanalytical column gradient and a Waters 515 isocratic pump for thesample digestion and loading. For sample digestion and loading, thebuffer used was 2% acetonitrile and 0.1% trifluoroacetic acid at a flowrate of 100 μL/min. For the analytical gradient, the buffers were BufferA) 0.1% formic acid in water and Buffer B) 0.1% formic acid inacetonitrile. The gradient was at 40 μL/min from 2% B to 36% B in 10minutes, followed by a wash of 80% B for 1.5 minute and are-equilibration at 2% B for 3 minutes. The column was then washed bycycling the gradient between 2% and 80% B, three times with 1 minute ateach step, followed by a final equilibration at 2% B for 5 minutes. Thetrapping column was a Waters VANGUARD™ C18 BEH 1.7 μm Guard Column andthe analytical column was a Waters C18 BEH300, 1.7 μm 1×50 mm column.

Sample handling for the deuterium labeling was done by a Leaptec H/D-XPAL™ system. The labeling sample tray was set to a temperature of 25°C., the quenching tray was set to 1.5 C and the trap and analyticalcolumn chamber was set to 1.5° C. The immobilized pepsin column(Pepsin/Protease XIII column NBA2014002, 2.1×30 mm, NovaBioAssay) waskept outside the column chamber at room temperature.

A deuterium labeling difference heatmap of the human ILT3-His amino acidresidues bound by the antibody is shown in FIG. 3A. The HDX massspectrometry shows that the antibody and the other antibody familiesdisclosed herein that cross-compete with the antibody bind an epitopecomprising or consisting of at least one amino acid in one or more ofamino acid residues 18-23 (ISWGNS; SEQ ID NO: 3), 64-69 (IPSMTE; SEQ IDNO: 4), 96-101 (MTGAYS; SEQ ID NO: 5), 124-131 (QSRSPMDT; SEQ ID NO: 6),152-159 (AQQHQAEF; SEQ ID NO: 7) and 184-187 (LLSH; SEQ ID NO: 8) ofILT3. FIG. 3B shows a first-view and a second view of athree-dimensional surface structure model of the human ILT3extracellular domain with the protected amino acid residues shown. Theseprotected amino acid residues comprise a split or non-contiguous epitopethat spans the border between the D1 and D2 domains of the extracellulardomain. FIG. 3C is a ribbon diagram showing the placement of the epitopeon the human ILT3 extracellular domain. Residues in black were protectedfrom labeling by the antibody. Residues in white showed no changes inlabeling and residues in dark gray did not have data acquired for them.The deuterium labeling difference for each residue was averaged andmapped onto a crystal structure of ILT3 (Cheng et al., “Crystalstructure of leukocyte Ig-like receptor LILRB4 (ILT3/LIR-5/CD85k): amyeloid inhibitory receptor involved in immune tolerance.” J Biol Chem286:18013-25 (2011)).

Similar HDX mapping experiments were preformed using antibodies ZM4.1,DX439, DX446, and 9B11. Antibody ZM4.1 is commercially available fromThermoFisher Scientific, Carlsbad, Calif. or BioLegend, San Diego,Calif. Antibodies DX439 and DX446 have been disclosed in WO2018089300and Antibody 9B11 has been disclosed in U.S. Pat. No. 7,777,008. Ofthese antibodies, only antibody ZM4.1 was observed to bind an epitopethat partially overlapped with the epitope bound by the antibodies ofthe present invention; however, binning studies showed that antibodyZM4.1 did not cross block binding of the antibodies of the presentinvention. FIGS. 3D, 3E, 3F, and 3G show heatmaps of the binding ofantibodies ZM4.1, DX439, DX446, and 9B11 to human ILT3.

Example 6 Pharmacokinetics of Chimeric Anti-ILT3 52B8 Mouse VH/HumanIGg4 (S228P):Mouse VL/Human Kappa (“c58B8”; mAb 73) in NSG Mice

The pharmacokinetics of chimeric anti-ILT3 52B8 mouse VH/human IgG4(S228P):mouse VL/human Kappa (c85B8; mAb 73) was evaluated in Panc08.13human-NSG mice model and SK-MEL-5 human CD34+-NSG mice model.

SK-MEL-5 is a human melanoma-derived line that can grow as asubcutaneous tumor. Panc 08.13 is a human pancreatic carcinoma-derivedtumor line. Panc 08.13 human-NSG model has been shown to be sensitive topembrolizumab and ipilimumab treatment. SK-MEL-5 model has a robust anddiverse myeloid infiltrate in the tumor compared to Panc 08.13 model.Both models show increased ILT3 expression on human CD14+ myeloid cellsin the tumor and spleen.

An ECL-based target capture immunoassay was used to quantify theantibody in humanized mice plasma. The assay was established withbiotinylated recombinant ILT3 as capture reagent, and sulfoTAG labeledmouse anti-huIgG (Fc specific) from Southern Biotech (cat #9190-01) fordetection reagent. Both calibrators and QCs were prepared in neatC57BL/6 plasma and diluted 100 times when testing in plate. This assayhas been qualified and the LLOQ of the assay was determined to be 40ng/mL with an MRD of 100.

In Panc08.13 hu-NSG mice model, 20 mg/kg of antibody was administeredwith and without pembrolizumab (5 mg/kg) via IP weekly for the firstthree doses and two weeks after the 3rd dose for the 4th dose. Bloodsamples were collected before the third dose (Ctrough) and 24 hoursafter the third dose (Cmax). Terminal blood samples on day 5 and 6 afterthe fourth dose were also collected. In SK-MEL-5 huCD34+-NSG mice model,the antibody was administered at 2 and 20 mg/kg via IP weekly. Bloodsamples were collected before the third dose (Ctrough) and 24 h afterthe third dose (Cmax). Terminal blood samples on day 3 and 7 after thethird dose were also collected. The free (unbound) antibodyconcentrations were determined by an antigen-capture assay.

Pharmacokinetic parameters are generated from historical IgG4 antibodydata (IV bolus administration of 1, 3, 10, 30 mg/kg of humanized IgG4antibody in C57BL/6J mice) with Phoenix NLME. PK profiles at the studieddose of the antibody were simulated based on the generatedpharmacokinetic parameters.

PK analysis of historical IgG4 antibody data showed a linearrelationship between AUC and studied dose (See FIG. 4). With theassumptions including linear PK across different tested doses of c52B8,no PK difference among different mouse strains, rapid absorption and100% bioavailable after IP administration of the antibody, PK profilesat the studied dose of c52B8 were simulated based on historical IgG4antibody data. The results showed that the simulated profile at 20 mg/kgin both Panc08.13 human-NSG model and SK-MEL-5 huCD34+-NSG model followthe observed c52B8 concentrations.

Example 7 Anti-ILT3 Monoclonal Antibodies Activate Dendritic Cells andReduces Suppressive Capacity of Myeloid-Derived Suppressor Cells (MDSCs)

Human PBMCs isolated from fresh leukopacs were frozen, thawed and CD14+monocytes were purified by negative selection. The purified cells werecultured for 5 days with GM-CSF (1000 U/mL) and IL4 (1000 U/mL). Theseimmature DCs were then further cultured for 42 hours with addition ofIL-10 (50 ng/mL) and LPS (1 ug/mL) with or without anti-ILT3 antibody.TNFα is measured in the culture supernatant.

Titration experiments showed that c52B8 caused a dose-dependent increasein TNFα secretion in the culture medium when added during thepolarization step, whereas a control IgG4 did not (the control is anvariant of a commercial antibody against RSV, trade name Synagis) (FIG.5A). The concentration of antibody required to produce half of themaximal increase in TNFα levels (EC50) was approximately 1.9 ng/mL. Thiswas not different for chimeric variants in which V_(H) and V_(L) ofp58B8 were fused to Fc with a human IgG1 framework (mAb 78) or a N297Amutated human IgG1 framework (mAb 76). These data indicate that in thisassay Fc receptor binding does not play any role in the functionalactivity. The independence from Fc receptor binding controls for thepossibility that the mechanism of activation in this assay is DCsbecoming activated through recognition of other DCs in the culture beingdecorated with antibody which would be a mechanism unrelated to ILT3.

FIGS. 5B and 5C show there was no significant difference in functionalactivity between c52B8 (mAb 73) and humanized anti-ILT3 mAb 52B8 VH1M64V/VL5 N34Q) IgG4 S228P/Kappa (mAb 46) in two donors. As shown, withantibody c52B8 added during polarization of the DCs, but not during Tcell priming, DCs were better able to activate T cells to proliferate,similar to DCs not tolerized with IL10. When antibody c52B8 was addedduring T cell priming but not during DC polarization, T cells werebetter able to respond to subsequent re-stimulation. Followinghumanization, variants that retained binding comparable to the chimerawere tested in this same assay and found to be active, with nomeaningful differences in potency among them. These data indicate thatdata generated with c52B8 is representative of what the data would be ifhumanized mAb 46 had been used.

Example 8 Anti-ILT3 Antibodies Reduce Suppressive Capacity ofMyeloid-Derived Suppressor Cells (MDSCs)

Without ascribing to any particular theory or hypothesis, we hypothesizethat a productive T cell response to tumor can be limited in some casesby the presence of immature and suppressive myeloid cells. These cellsexpress ILT3 and we hypothesize that ILT3 functions as an inhibitorymanner to maintain an immature state characterized by low HLA-DRexpression, IL-10 production, and effective suppression of T cellactivation and proliferation. Establishment of a model based onco-culture of human PBMCs with SKMEL5 tumor cells in vitro, followed bypurification of MDSCs and testing of their ability to suppressproliferation of autologous CD8+ T cells enabled exploration of thisaspect of ILT3 biology. This example shows that c52B8 and humanized 52B8(mAb 46) are able to impair the acquisition (or maintenance) of a Tcell-suppressive phenotype.

To generate MDSCs, healthy human PBMCs were cultured with SKMEL5 cellsand 20 ng/mL GM-CSF for 7 days. CD33+ cells were collected by positiveantibody-based magnetic bead selection and then co-cultured at theindicated ratios with purified autologous CD8+ T cells for 3 days in thepresence of a polyclonal stimulus. Cultures included c52B8 (mAb 73),humanized 52B8 (mAb 46), or isotype control antibody (1 μg/mL) in boththe co-culture and T cell suppression steps. The T cell suppressionassay was conducted with a T cell to MDSC ratio of 4:1 and measuring theamount of interferon gamma (INFγ) produced.

FIG. 6A and FIG. 6B exemplifies the activity of both humanized 52B8 andc52B8 in the MDSC model at a ratio of T cells to MDSCs where the effectof these antibodies was most evident show that the antibodies reduce thesuppressive capacity of MDSCs in a comparable manner. These data furtherindicate that data generated with c52B8 is representative of what wouldbe found with humanized mAb 46.

Example 9 Anti-ILT3 Antibody cC52B8 Inhibits Growth of SK-MEL-5 Tumorsin SK-MEL-5 hu-NSG Mice Bearing SK-MEL-5 Subcutaneous Tumors

Systemic administration of c52B8 once weekly to mice bearing establishedsubcutaneous tumors afforded inhibition of tumor growth (FIG. 7).Animals were randomized to treatment on the basis of tumor volume on day21 post-implantation and dosed s.c. with 20 mg/kg of c52B8 (mAb 73) orisotype control once weekly beginning on day 21. Data shown in the leftpanel are means and std. error (nine per group). Individual animal tumorgrowth curves are shown at right. Body weight decreased to a similardegree in both control and 52B8 groups. This study is representative ofthree independent studies.

The degree of inhibition of tumor growth was consistent and similar inthree separate studies and was very similar to the effect of anti-ILT4.None of the other mechanisms tested to date (e.g. anti-PD-1, anti-ILT4,anti-CD27, anti-GITR) have afforded regressions leading us to speculatethat tumor stasis may represent a floor for this model. This is clearlydifferent from the mouse syngenic models commonly used for preclinicalefficacy assays.

Example 10 Immune Activation in SK-MEL-5 hu-NSG after c52B8 Treatment

To understand immune mechanism that mediates the tumor efficacy, tumorinfiltrating immune cells were profiled and measured sHLA-G levels weremeasured in the blood. Mice were treated with c52B8 (2 and 20 mg/kg i.p.QW). Antibody doses were selected based on C_(max) and C_(trough) levelsdetected in a mini-PK and simulations using historical studies. Bloodsamples were collected for PK, sHLA-G, and cytokine analyses. TILsprofiling was performed using CyTOF to detect 36 markers simultaneously.Terminal tumor samples were fixed and used for human CD3+ T cell IHCanalysis. Thirty percent tumor growth inhibition was observed in micetreated with 20 mpk 52B8. However, no statistical significant differencewas detected due to big variability associated with the humanized tumormodel. 52B8 modest tumor efficacy was associated with a modest decreasein tumor CD4+CD127−CD25+ T suppressor cells (21% vs. 14%) and bloodsHLA-G levels and an increase in activation of T cells (CD69 intensity,14 vs. 23) in the tumor. No cytokine change was detected with c52B8treatment as seen in FIG. 8.

Example 11 Effect of Anti-ILT3 Antibody c52B8 in Combination withPembrolizumab in Panc 08.13 hu-NSG Model: Tumor Efficacy and ImmuneActivation

Anti-ILT3 antibody c52B8 was evaluated in Panc 08.13 hu-NSG model. 52B8used as a single agent showed minimum effect on tumor growth inhibition.When 52B8 was used in combination with pembrolizumab, one in fivecohorts (five different human donors) of humanized mice had 50% tumorgrowth inhibition (TGI) and the TGI was associated with increased T cellactivation and IFNγ production and decreased blood sHLA-G level as seenin FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D.

Example 12 Effect of Anti-ILT3 Antibody 52B8 in Combination withPembrolizumab in an MDSC/T Cell Suppression Assay

Humanized anti-ILT3 antibody 52B8 (mAb 46) with and withoutpembrolizumab effected an increase T-Cell activity in MDSC/T-cellsuppression assays. The effect was additive when mAb 46 was used incombination with pembrolizumab.

To generate MDSCs, healthy human PBMCs from a particular donor werecultured with SKMEL5 cells and 20 ng/mL GM-CSF for seven days. Cultureswere treated with 52B8 (1 μg/mL) or isotype control antibody (1 μg/mL).CD33+ cells were collected anti-CD33 magnetic microbeads and LS columnseparation (Miltenyi Biotec, Germany) and then co-cultured at theindicated ratios with purified autologous CD8+ T cells for 3 days in thepresence of a polyclonal stimulus. Autologous CD8+ T cells were isolatedfrom healthy human PBMCs using negative antibody-based magnetic beadselection (Stem Cell Technologies, Canada) then co-cultured in 96 wellplates with CD33+ myeloid cells at the ratio of 8:1 (Tcell:MDSC) for 2days. Cultures included humanized 52B8 (mAb 46) or isotype controlantibody (IgG4) (1 μg/mL) alone or in combination with pembrolizumab (2μg/mL) in both the co-culture and T cell suppression steps. Totalantibody concentration in each treatment is adjusted to 3 ug/mL withisotype control antibody. T cell proliferation was induced by apolyclonal stimulus anti-CD3/CD28 beads and IL2. IFNγ levels weredetermined in culture supernatants using MSD ELISA (Mesoscale Discovery,Md.). The T cell suppression assay was conducted with a T cell to MDSCratio of 4:1 or 8:1 and measuring the amount of interferon gamma (INFγ)produced. The results are shown in FIGS. 10-14.

FIG. 10 shows that humanized anti-ILT3 antibody 52B8 (mAb 46) reducesthe suppressive capacity of MDSCs to an extent comparable to chimericanti-ILT3 antibody c52B8 (mAb 73; lot 26AVY) in MDSC/T-cell suppressionassays using MDSCs obtained from PBMCs from two different human donors(D00100385 and D001003507, respectively).

As shown in FIGS. 11-14 humanized anti-ILT3 antibody 52B8 (mAb 46) incombination with pembrolizumab reduced MDSC inhibition of T cellactivation at a higher level compared to either alone in an MDSC/T cellsuppression assay (a) at either a 4:1 or 8:1 ratio of T cell to MDSCusing MDSCs obtained from PBMCs from human donor D001003835 (FIG. 11);(b) at either a 4:1 or 8:1 ratio of MDSC to T cell using MDSCs obtainedfrom PBMCs from human donor D001003180 (FIG. 12); (c) at a 4:1 or 8:1ratio of ratio of T cell to MDSC using MDSCs obtained from PBMCs fromhuman donor D001003507 (FIG. 13); and an 8:1 ratio of ratio of T cell toMDSC using MDSCs obtained from PBMCs from human donor (FIG. 14). Theresults are summarized in Tables 8 and 9. As shown in FIGS. 10-13 andTables 8 and 9, combining an anti-ILT3 antibody 52B8 with pembrolizumabresulted in an additive effect of increasing the activation of T cellsover that achievable using pembrolizumab or 52B8 alone. As shown,increases in IFNγ for the combination relative to the other treatmentsranged from 41% to 74%. These results indicate that the combination ofpembrolizumab with 52B8 does not result in an excessive or uncontrolledescalation of T cell activation.

TABLE 8 Summary of the humanized anti-ILT3 antibody 52B8 andpembrolizumab combination data Mean Avg ± SD T cell + MDSC T Cell:hIgG4 + 52B8 + MDSC T cell hIgG4 + Pembro- hIgG4 + Pembro- Donor ratioonly hIgG4 lizumab 52B8 lizumab D001003835 4:1  19439 ±  3667 ±  4676 ± 6380 ± 10438 ±  4191 795 1162 1187 1132 8:1  32644 ± 17386 ± 20556 ±28280 ± 38163 ±  4146 1628 5028 4643 7817 D001003180 4:1  38166 ±  1482±  1781 ±  3983 ±  3606 ±  7574 646 295 1528 1864 8:1  33250 ±  6823 ± 6768 ± 9532 ± 3025 14896 ±  6021 2107 1287 2932 D001003507 4:1  56836 ± 7364 ±  8111 ± 12202 ± 18422 ±  5777 2977 5220 3221 4135 8:1  55376 ±23417 ± 23981 ± 26204 ± 36992 ±  6310 8640 3135 3075 1856 D001003428 8:1159127 ± 81071 ± 87413 ± 98902 ± 123920 ±  10552 13458 15061 8994 22448 

TABLE 9 52B8 Antibody + Pembrolizumab Combination- T cell: MDSC ratio(8:1) Ratios of Condition GM 95% CI P-value (52B8 + pembrolizumab)/ 1.841.35, 2.53 0.0043 IgG4 (52B8 + pembrolizumab)/ 1.73 1.26, 2.36 0.0057pembrolizumab (52B8 + pembrolizumab)/ 1.39 1.20, 1.61 0.0028 52B8 Thep-values are from one-sided paired t-tests comparing the 52B8 +pembrolizumab combination to each of the other groups, using logs ofIFNγ values. GM = geometric mean

Example 13 Effect of Anti-ILT3 Antibody 52B8 in Combination withPembrolizumab in Mixed Lymphocyte Reaction of Polarized IL-10 DCs andAllogenic CD8+ T Cells

In this example, a mixed lymphocyte reaction of IL-10-polarized humanmonocyte-derived dendritic cells and allogenic CD8+ T cells, incubatedfor four days followed by measurement of interferon gamma (IFNγ) in theculture supernatant as a read out of T cell activation. In thisexperiment, the activities of pembrolizumab, 52B8, or the combination ofthe two were compared to isotype control antibody (IgG4 in both cases),in nine allogenic donor pairs.

Monocyte derived dendritic cells (DCs)—IL10 DCs from three CD14+monocyte donors were differentiated for seven days(Granulocyte-macrophage colony-stimulating factor (GMCSF) and IL4 forfive days and then two days with IL10, with and without IgG4 (lot92ASJ), with and without 52B8 (Lot 41BAB) at 1 μg/mL) to produce DC129,DC226, and DC196. CD8+ cells from three donors were isolated and mixedleukocyte reactions (MLR) were established at 1:5 DC:T cell ratio fromthe three donors in a 96 well format (30 k DC vs 150 k CD8+ T cells)where cells were treated with and without IgG4 (lot 92 ASJ); with andwithout Pembrolizumab (lot 42ASN) at 2 μg/mL. IgG4 or 52B8 was alsoadded back in the MLR at 1 μg/mL. Wound up with nine MLR pairs of IL10DCs:CD8+ T cells:

-   -   DC129 vs T30, T3788 and T3259    -   DC226 vs T30, T3788 and T3259    -   DC196 vs T30, T3788 and T3259        IFNγ supernatant was collected at day four and quantified using        Meso Scale Discovery (MSD). Additional supernatant fraction was        collected at day five and cells were collected and stained for        PD1 and PDL1 expression. Dendritic Cell Staining on Day seven of        differentiation (just prior to MLR setup). T cell Staining of        CD8+ T cells at day five of MLR assay.

FIG. 15 shows the results for all donor pairs combined into one figure(each mark is a donor pairing). As shown, 52B8 in combination withpembrolizumab effected a reverse of T cell tolerization, resulting in astatistically significant increase in activation of CD8+ T cells.

Table of Sequences SEQ ID NO: Description Sequence 1 Human ILT3QAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDK (LILRB4)EESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSP extracellularVGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSGKSVTLLC domain with C-QSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPV terminal His Tag;TSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSP epitope domainsTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWEHHHHHHHH identified by bold- face type 2Macaca mulatta QAGPLPKPTIWAEPGSVISWGSPVTIWCQGTLDAQEYYLDKE (Rhesus) ILT3GSPAPWDTQNPLEPRNKAKFSIPSMTQHYAGRYRCYYHSHP (LILRB4)DWSEDSDPLDLVMTGAYSKPILSVLPSPLVTSGESVTLLCQS extracellularQSPMDTFLLFKEGAAHPLPRLRSQHGAQLHWAEFPMGPVTS domainVHGGTYRCISSRSFSHYLLSRPSDPVELTVLGSLESPSPSPTRSI (sequence obtainedSAAGPEDQSLMPTGSDPQSGLRRHWE from GenBank NP_001035766) 3 Human ILT3ISWGNS peptide A 4 Human ILT3 IPSMTE peptide B 5 Human ILT3 MTGAYSpeptide C 6 Human ILT3 QSRSPMDT peptide D 7 Human ILT3 AQQHQAEFpeptide E 8 Human ILT3 LLSH peptide F 9 Human IgG4 HCASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA Constant domainLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN (S228P; shown inTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR bold-face type)TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 10Human IgG4 HC ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAConstant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN(S228P; shown in TKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR bold-face type)TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS (lacks C-terminal KTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP (herein referred toREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP as “K-”))ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 11Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAconstant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 12Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAConstant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN(L234A, L235A, TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLD265S; shown in MISRTPEVTCVVVSV

HEDPEVKFNWYVDGVEVHNAKTKPREEQ bold-face type)YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 13Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAConstant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN(K-) (L234A, TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL L235A, D265S;MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ shown in bold-faceYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK type)GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 14Human LC Kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDConstant domain NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 15 Anti-ILT3 52B8EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP parental HCDRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ variable domainMSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS 16 Anti-ILT3 52B8NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQQ parental LCKPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPVEAD variable domainDAATYYCQQNNEDPYTFGGGTKLEIK 17 52B8 HC-CDR1 NYGMS 18 52B8 HC-CDR2TISGGGDYTNYPDSXRG (Wherein Xaa15 is M, V, or L) 19 52B8 HC-CDR2 MTISGGGDYTNYPDSMRG 20 52B8 HC-CDR2 V TISGGGDYTNYPDSVRG 21 52B8 HC-CDR2 LTISGGGDYTNYPDSLRG 22 52B8 HC-CDR3 RLXFRSLYYAMDY (Wherein Xaa3 isW, Y, Q, or F) 23 52B8 HC-CDR3 RLWFRSLYYAMDY 24 52B8 HC-CDR3RLYFRSLYYAMDY 25 52B8 HC-CDR3 RLQFRSLYYAMDY 26 52B8 HC-CDR3RLFFRSLYYAMDY 27 52B8 LC-CDR1 RASEKVDSFGXXFMH (Wherein Xaa11 isN, D, or Q and Xaa12 is S, N, or A) 28 52B8 LC-CDR1 N RASEKVDSFGNXFMH(Wherein Xaa12 is S, N, or A) 29 52B8 LC-CDR1 D RASEKVDSFGDXFMH(Wherein Xaa12 is S, N, or A) 30 52B8 LC-CDR1 Q RASEKVDSFGQXFMH(Wherein Xaa12 is S, N, or A) 31 52B8 LC-CDR1 S RASEKVDSFGXSFMH(Wherein Xaa11 is N, D, or Q) 32 52B8 LC-CDR1 N RASEKVDSFGXNFMH(Wherein Xaa11 is N, D, or Q) 33 52B8 LC-CDR1 A RASEKVDSFGXAFMH(Wherein Xaa11 is N, D, or Q) 34 52B8 LC-CDR1 RASEKVDSFGNNFMH (NN) 3552B8 LC-CDR1 RASEKVDSFGDNFMH (DN) 36 52B8 LC-CDR1 RASEKVDSFGQNFMH (QN)37 52B8 LC-CDR1 RASEKVDSFGNSFMH (NS) 38 52B8 LC-CDR1 RASEKVDSFGDSFMH(DS) 39 52B8 LC-CDR1 RASEKVDSFGNAFMH (NA) 40 52B8 LC-CDR1RASEKVDSFGDAFMH (DA) 41 52B8 LC-CDR1 RASEKVDSFGQSFMH (QS) 4252B8 LC-CDR1 RASEKVDSFGQAFMH (AF) 43 52B8 LC-CDR2 LTSNLDS 4452B8 LC-CDR3 QQNNEDPYT 45 Anti-ILT3 40A6QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG parental HCKGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN variable domainSLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSS 46 Anti-ILT3 40A6ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG parental LCQSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISDVEPEDLG variable domainVYYCLQYGSVPYTFGAGTKLELK 47 40A6 HC-CDR1 SYSIN 48 40A6 HC-CDR2RFWYDEGIAYNLTLES 49 40A6 HC-CDR3 DRDTVGITGWFAY 50 40A6 LC-CDR1KASQSVGVNVD 51 40A6 LC-CDR2 GSANRHT 52 40A6 LC-CDR3 LQYGSVPYT 53Anti-ILT3 16B1 QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS parental HCGKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM variable domainNSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSS 54 Anti-ILT3 16B1ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ parental LCSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLGV variable domainYYCLQYGSVPYTFGPGTKLELK 55 16B1 HC-CDR1 NYCVN 56 16B1 HC-CDR2RFWFDEGKAYNLTLES 57 16B1 HC-CDR3 DRDTVGITGWFAY 58 16B1 LC-CDR1KASQSVGINVD 59 16B1 LC-CDR2 GSANRHT 60 16B1 LC-CDR3 LQYGSVPYT 61Anti-ILT3 11D1 QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP parental HCGHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ variable domainLSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSS 62 Anti-ILT3 11D1DIQMTQSPSSLSVSLGGKVTITCKASQDINEYIGWYQRKPGK parental LCGPRLLIHYTSTLQSGIPSRFSGSGSGRDYSLSISNLEPEDIATYY variable domainCLQYANPLPTFGGGTKLEIK 63 11D1 HC-CDR1 TYWIE 64 11D1 HC-CDR2EILPGNGNTHFNENFKD 65 11D1 HC-CDR3 RRLGRGPFDF 66 11D1 LC-CDR1 KASQDINEYIG67 11D1 LC-CDR2 YTSTLQS 68 11D1 LC-CDR3 LQYANPLPT 69 Anti-ILT3 17H12EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQA parental HCPTRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ variable domainMDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSS 70 Anti-ILT3 17H12DIVLTQSPALAVSLGQRATISCRASQSVSMSRYDLIHWYQQK parental LCPGQQPKLLIFRASDLASGIPARFSGSGSGTDFTLTINPVQADDI variable domainATYYCQQTRKSPPTFGGGTRLELK 71 17H12 HC-CDR1 NFDMA 72 17H12 HC-CDR2SITYDGGSTSYRDSVKG 73 17H12 HC-CDR3 VESIATISTYFDY 74 17H12 LC-CDR1RASQSVSMSRYDLIH 75 17H12 LC-CDR2 RASDLAS 76 17H12 LC-CDR3 QQTRKSPPT 77Anti-ILT3 37C8 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG parental HCKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN variable domainRLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSS 78 Anti-ILT3 37C8ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ parental LCSPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV variable domainYYCLQYGSVPYTFGPGTKLELK 79 37C8 HC-CDR1 SYCVN 80 37C8 HC-CDR2RFWYDEGKVYNLTLES 81 37C8 HC-CDR3 DRDTMGITGWFAY 82 37C8 LC-CDR1KASQSVGINVD 83 37C8 LC-CDR2 GSANRHT 84 37C8 LC-CDR3 LQYGSVPYT 85Anti-ILT3 1G12 QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP parental HCGHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIHLS variable domainSLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSS 86 Anti-ILT3 1G12DIQMTQSPSSLSASLGGKVTITCEASQDINKHIDWYQHQPGR parental LCGPSLLIHYASILQPGIPSRFSGSGSGRDYSFSITSLEPEDIATYY variable domainCLQYDNLLPTFGGGTKLEIK 87 1G12 HC-CDR1 TYWIQ 88 1G12 HC-CDR2EILPGSGTTNYNENFKG 89 1G12 HC-CDR3 RLGRGPFDY 90 1G12 LC-CDR1 EASQDINKHID91 1G12 LC-CDR2 YASILQP 92 1G12 LC-CDR3 LQYDNLLPT 93 Anti-ILT3 20E4QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPSG parental HCKGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM variable domainNSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSP 94 Anti-ILT3 20E4ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG parental LCQSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLG variable domainVYYCLQYGSVPYTFGAGTKLELK 95 20E4 HC-CDR1 SYSVN 96 20E4 HC-CDR2RFWYDGGTAYNSTLES 97 20E4 HC-CDR3 DRDTMGITGWFAY 98 20E4 LC-CDR1KASQSVGVNVD 99 20E4 LC-CDR2 GSANRHT 100 20E4 LC-CDR3 LQYGSVPYT 101Anti-ILT3 24A4 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG parental HCKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN variable domainRLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSS 102 Anti-ILT3 24A4ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ parental LCSPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV variable domainYYCLQYGSVPYTFGPGTKLELK 103 24A4 HC-CDR1 SYCVN 104 24A4 HC-CDR2RFWYDEGKVYNLTLES 105 24A4 HC-CDR3 DRDTLGITGWFAY 106 24A4 LC-CDR1KASQSVGINVD 107 24A4 LC-CDR2 GSANRHT 108 24A4 LC-CDR3 LQYGSVPYT 109Leader sequence A MEWSWVFLFFLSVTTGVHS 110 Leader sequence BMSVPTQVLGLLLLWLTDARC 111 Mouse Anti-ILT3EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP p52B8 parental HC:DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ Murine IgG2aMSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS heavy chainAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 112 Mouse Anti-ILT3NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQQ p52B8 parental LC:KPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPVEAD murine Kappa lightDAATYYCQQNNEDPYTFGGGTKLEIKRADAAPTVSIFPPSSE chainQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC 113 Chimeric Anti-EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP ILT3 mouse 52B8DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ VH parental/humanMSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS IgG4 (S228P)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 114Chimeric Anti- EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP ILT3 mouse 52B8DRRLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNTLYLQ VH M64V/humanMSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS IgG4 (S228P)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 115Mouse Anti-ILT3 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP 52B8 VHDRRLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNTLYLQ M64L/human IgG4MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS (S228P)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 116Chimeric Anti- NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQQILT3 mouse 52B8 KPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPVEAD parental VL/DAATYYCQQNNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ human KappaLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C 117 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS S 118 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V) S 119 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L) S 120 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F) 121Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y) 122Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q) 123Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS S 124 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V) S 125 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L) S 126 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSRTDFTLTISSLQAED domain VL1VAVYYCQQNNEDPYTFGQGTKLEIK 127 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2VAVYYCQQNNEDPYTFGQGTKLEIK 128 Humanized 52B8EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variableKPGQAPRLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLEPED domain VL3FAVYYCQQNNEDPYTFGQGTKLEIK 129 Humanized 52B8EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variableKPGQAPRLLIYLTSNLDSGIPARFSGSGSGTDFTLTISSLEPEDF domain VL4AVYYCQQNNEDPYTFGQGTKLEIK 130 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5FATYYCQQNNEDPYTFGQGTKLEIK 131 Humanized 52B8DIQMTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL6FATYYCQQNNEDPYTFGQGTKLEIK 132 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL7FATYYCQQNNEDPYTFGQGTKLEIK 133 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLQPED domain VL8FATYYCQQNNEDPYTFGQGTKLEIK 134 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNAFMHWYQ LC variableQKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAE domain VL2,DVAVYYCQQNNEDPYTFGQGTKLEIK (S35A) 135 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNNFMHWYQ LC variableQKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAE domain VL2,DVAVYYCQQNNEDPYTFGQGTKLEIK (S35N) 136 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGQSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2,VAVYYCQQNNEDPYTFGQGTKLEIK (N34Q) 137 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGDSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2,VAVYYCQQNNEDPYTFGQGTKLEIK (N34D) 138 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNAFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5,FATYYCQQNNEDPYTFGQGTKLEIK (S35A) 139 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNNFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5,FATYYCQQNNEDPYTFGQGTKLEIK (S35N) 140 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5FATYYCQQNNEDPYTFGQGTKLEIK (N34Q) 141 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGDSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5,FATYYCQQNNEDPYTFGQGTKLEIK (N34D) 142 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domainMNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS VH1/Human IgG4SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) constantALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS domain NTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 143Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 144Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 145Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V,ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101F)/HumanLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P)TKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 146Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V,ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101Y)/HumanLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P)TKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 147Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V,ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101Q)/HumanLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P)TKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 148Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domainMNSLKAEDTAVYYCGRRLWFRSLYVAMDYWGQGTLVTVS VH2/Human IgG4SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) constantALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS domain NTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 149Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 150Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 151Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSRTDFTLTISSLQAED domain VL1/kappaVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 152 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2/kappaVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 153 Humanized 52B8EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variableKPGQAPRLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLEPED domain VL3/kappaFAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 154 Humanized 52B8EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variableKPGQAPRLLIYLTSNLDSGIPARFSGSGSGTDFTLTISSLEPEDF domain VL4/kappaAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLK constant domainSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 155 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5/kappaFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 156 Humanized 52B8DIQMTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL6/kappaFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 157 Humanized 52B8DIQLTQ SPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL7/kappaFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 158 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLQPED domain VL8/kappaFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domainKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 159 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNAFMHWYQ LC variableQKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAE domain VL2DVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQ (S35A)/kappaLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS constant domainKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C 160 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNNFMHWYQ LC variableQKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAE domain VL2DVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQ (S35N)/kappaLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS constant domainKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C 161 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGQSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2VAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34Q)/kappaKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domainDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 Humanized 52B8DIVLTQSPDSLAVSLGERATINCRASEKVDSFGDSFMHWYQQ LC variableKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2VAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34D)/kappaKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domainDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 163 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNAFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (S35A)/kappaKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domainDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 164 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNNFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (S35N)/kappaKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domainDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 165 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34Q)/kappaKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domainDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 166 Humanized 52B8DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGDSFMHWYQQ LC variableKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34D)/kappaKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domainDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 167 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1/MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS Human IgG1 HCSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A L235AALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) constantNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT domain LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 168Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) constant LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 169Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) constant LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 170Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F)/ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,TKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTL D265S) constant MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 171Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y)/ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,TKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTL D265S) constant MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 172Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q)/ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,TKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTL D265S) constant MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 173Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2/MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS Human IgG1 HCSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A, L235A,ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) constantNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT domain LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 174Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) constant LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 175Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) constant LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 176Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domainMNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS VH1/Human IgG4SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) (K-)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 177Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 178Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 179Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V),ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101F/HumanLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P) (K-)TKVDKRVESKYGP

CPPCPAPEFLGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 180Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V,ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101Y)/HumanLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P) (K-)TKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 181Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V,ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 101Q)/Human IgG4LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN (S228P) (K-)TKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 182Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domainMNSLKAEDTAVYYCGRRLWFRSLYVAMDYWGQGTLVTVS VH2/Human IgG4SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) (K-)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 183Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYVAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 184Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/HumanSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domainNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 185Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1/MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS Human IgG1 HCSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A, L235A,ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) (K-)NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT constant domain LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 186Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/ HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) (K-) LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 187Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L)/ HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) (K-) LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 188Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F)/ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,TKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTL D265S) (K-) MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ constant domainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 189Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y)/ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,TKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTL D265S) (K-) MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ constant domainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 190Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q)/ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HCLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,TKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDTL D265S) (K-) MISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ constant domainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 191Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2/MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS Human IgG1 HCSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A, L235A,ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) (K-)NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT constant domain LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 192Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS M64V/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) (K-) LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 193Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS M64L/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,NTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPKPKDT D265S) (K-) LMISRTPEVTCVVV

VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domainQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 194Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG ILT3 rat 40A6KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN parental HCSLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAST variableKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS domain/humanGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV IgG4 (S228P)DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 195Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG ILT3 rat 40A6QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISDVEPEDLG parental LCVYYCLQYGSVPYTFGAGTKLELKRTVAAPSVFIFPPSDEQLKS variableGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS domain/humanTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 196 Chimeric Anti-QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS ILT3 rat 16B1GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM parental HCNSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS variableTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT domain/humanSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK IgG4 (S228P)VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS constant domainRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 197Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ ILT3 rat 16B1SPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLGV parental LCYYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGT variableASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY domain/humanSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 198 Chimeric Anti-QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP ILT3 mouse 11D1GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ parental HCLSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSSASTKGP variableSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH domain/humanTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK IgG4 (S228P)VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE constant domainVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 199Chimeric Anti- DIQMTQSPSSLSVSLGGKVTITCKASQDINEYIGWYQRKPGKILT3 mouse 11D1 GPRLLIHYTSTLQSGIPSRFSGSGSGRDYSLSISNLEPEDIATYYparental LC CLQYANPLPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS variableVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL domain/humanSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 200 Chimeric Anti-EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQA ILT3 rat 17H12PTRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ parental HCMDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSSAS variableTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT domain/humanSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK IgG4 (S228P)VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS constant domainRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 201Chimeric Anti- DIVLTQSPALAVSLGQRATISCRASQSVSMSRYDLIHWYQQK ILT3 rat 17H12PGQQPKLLIFRASDLASGIPARFSGSGSGTDFTLTINPVQADDI parental LCATYYCQQTRKSPPTFGGGTRLELKRTVAAPSVFIFPPSDEQLKS variableGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS domain/humanTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 202 Chimeric Anti-QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG ILT3 rat 37C8KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN parental HCRLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSSAST variableKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS domain/humanGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV IgG4 (S228P)DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 203Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ ILT3 rat 37C8SPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV parental LCYYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGT variableASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY domain/humanSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 204 Chimeric Anti-QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP ILT3 mouse 1G12GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIHLS parental HCSLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSSASTKGPSV variableFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF domain/humanPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE IgG4 (S228P)PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT constant domainCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 205Chimeric Anti- DIQMTQSPSSLSASLGGKVTITCEASQDINKHIDWYQHQPGRILT3 mouse 1G12 GPSLLIHYASILQPGIPSRFSGSGSGRDYSFSITSLEPEDIATYYparental LC CLQYDNLLPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS variableVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL domain/humanSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 206 Chimeric Anti-QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPSG ILT3 rat 20E4KGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM parental HCNSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSPAS variableTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT domain/humanSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK IgG4 (S228P)VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS constant domainRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 207Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG ILT3 rat 20E4QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLG parental LCVYYCLQYGSVPYTFGAGTKLELKRTVAAPSVFIFPPSDEQLKS variableGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS domain/humanTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 208 Chimeric Anti-QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG ILT3 rat 24A4KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN parental HCRLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSSAST variableKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS domain/humanGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV IgG4 (S228P)DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR constant domainTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 209Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ ILT3 rat 24A4SPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV parental LCYYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGT variableASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY domain/humanSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 210 Humanized 52B8EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variableGKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V)/HumanSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HCALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (N297A) constantNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL domainMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 211Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAconstant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN(N297A; shown in TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLbold-face type) MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 212Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG40A6 rat VH/ KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN human IgG1SLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAST (N297A)KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 213Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS16B1 rat VH/ GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM human IgG1NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS (N297A)TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 214Chimeric anti-ILT3 QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP11D1 mouse VH/ GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ human IgG1LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSSASTKGP (N297A)SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 215Chimeric anti-ILT3 EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQA17H12 rat VH/ PTRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ human IgG1MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSSAS (N297A)TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 216Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG37C8 rat VH/ KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN human IgG1RLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSSAST (N297A)KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 217Chimeric anti-ILT3 QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP1G12 mouse VH/ GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIHLS human IgG1SLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSSASTKGPSV (N297A)FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 218Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPSG20E4 rat VH/ KGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM human IgG1NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSPAS (N297A)TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 219Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG24A4 rat VH/ KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN human IgG1RLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSSAST (N297A)KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 220Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG40A6 rat VH/ KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN human IgG1SLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAST (N297A)KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 221Residues after LC- FGXG CDR3 Xaa is any amino acid 222 Residues beforeCXXX HC-CDR1 Xaa is any amino acid 223 Residues before LEWIG HC-CDR1 224Residues after HC- WGXG CDR3 Xaa is any residue 225 PembrolizumabQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQ Heavy ChainAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYVVGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 226 PembrolizumabEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQ Light ChainKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 227 Human IgG1 HCASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA constant domainLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (N297A, D265A;TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL shown in bold-faceMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREE type)QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDL4VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKConstant regions are shown in italics. Amino acid sequences underlinedare CDRs.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1.-18. (canceled)
 19. A chimeric, humanized, or recombinant humanantibody or antigen binding fragment that cross-blocks or competes withthe binding of an antibody comprising a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO: 15 and a light chain comprisingthe amino acid sequence shown in SEQ ID NO: 16 from binding to humanimmunoglobulin-like transcript 3 (ILT3). 20.-30. (canceled)
 31. Thechimeric, humanized, or recombinant human antibody or antigen bindingfragment of claim 19, wherein the chimeric, humanized, or recombinanthuman antibody or antigen binding fragment binds to an epitope on humanILT3 comprising the amino acid sequences set forth in SEQ ID NOs: 3, 4,5, 6, 7, and 8 as determined by hydrogen deuterium exchange massspectrometry (HDX-MS) analysis.
 32. The chimeric, humanized, orrecombinant human antibody or antigen binding fragment of claim 31,wherein the epitope consists of the amino acid sequences set forth inSEQ ID NOs: 3, 4, 5, 6, 7, and
 8. 33. The chimeric, humanized, orrecombinant human antibody or antigen binding fragment of claim 19 thatcomprises at least two heavy chains (HCs) and two light chains (LCs)inter-connected by disulfide bonds.
 34. The chimeric, humanized, orrecombinant human antibody or antigen binding fragment of claim 19 thathas a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹¹ M.
 35. Thechimeric, humanized, or recombinant human antibody or antigen bindingfragment of claim 34 that comprises at least two heavy chains (HCs) andtwo light chains (LCs) inter-connected by disulfide bonds.
 36. Thechimeric, humanized, or recombinant human antibody or antigen bindingfragment of claim 19 that has a dissociation constant (K_(D)) of 5×10⁻⁹M or less.
 37. The chimeric, humanized, or recombinant human antibody orantigen binding fragment of claim 36 that comprises at least two heavychains (HCs) and two light chains (LCs) inter-connected by disulfidebonds.
 38. The chimeric, humanized, or recombinant human antibody orantigen binding fragment of claim 19 that does not bind with measurablebinding to human ILT5, human ILT7, human ILT8, and human ILT11 asdetermined in a cell enzyme linked immunosorbent assay (ELISA) orBiacore assay using 10 μg/mL of the chimeric, humanized, or recombinanthuman antibody or antigen binding fragment.
 39. The chimeric, humanized,or recombinant human antibody or antigen binding fragment of claim 38that comprises at least two heavy chains (HCs) and two light chains(LCs) inter-connected by disulfide bonds.
 40. The chimeric, humanized,or recombinant human antibody or antigen binding fragment of claim 19that is a variant of an antibody or antigen binding fragment comprising(a) a heavy chain complementarity determining region (HC-CDR) 1, whereinthe HC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 17; anHC-CDR2, wherein the HC-CDR2 has the amino acid sequence set forth inSEQ ID NO: 19, 20, or 21; an HC-CDR3, wherein the HC-CDR3 has the aminoacid sequence set forth in SEQ ID NO: 23; and (b) a light chaincomplementarity determining region (LC-CDR) 1, wherein the LC-CDR1 hasthe amino acid sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39,40, 41, or 42; an LC-CDR2, wherein the LC-CDR2 has the amino acidsequence set forth in SEQ ID NO: 43; and an LC-CDR3, wherein the LC-CDR3has the amino acid sequence set forth in SEQ ID NO: 44; wherein thevariation is one, two, or three amino acid substitutions, additions,deletions, or combinations thereof in one or more of the HC-CDRs orLC-CDRs.
 41. The chimeric, humanized, or recombinant human antibody orantigen binding fragment of claim 40 wherein the variation is one, two,or three conservative amino acid substitutions.